WO2018179514A1 - Dispositif de détection de composé contenant un squelette stéroïde et procédé de détection de composé contenant un squelette stéroïde l'utilisant - Google Patents

Dispositif de détection de composé contenant un squelette stéroïde et procédé de détection de composé contenant un squelette stéroïde l'utilisant Download PDF

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WO2018179514A1
WO2018179514A1 PCT/JP2017/034996 JP2017034996W WO2018179514A1 WO 2018179514 A1 WO2018179514 A1 WO 2018179514A1 JP 2017034996 W JP2017034996 W JP 2017034996W WO 2018179514 A1 WO2018179514 A1 WO 2018179514A1
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region
dimensional
nucleic acid
formation
stem
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PCT/JP2017/034996
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Japanese (ja)
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金子　直人
克紀 堀井
大橋　啓之
逢坂　哲彌
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Ｎｅｃソリューションイノベータ株式会社
学校法人早稲田大学
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Priority to JP2019508523A priority Critical patent/JP6823244B2/ja
Publication of WO2018179514A1 publication Critical patent/WO2018179514A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS

Definitions

  • the present invention relates to a steroid skeleton-containing compound detection device and a steroid skeleton-containing compound detection method using the device.
  • Detecting targets is required in various fields such as clinical medicine, food, and environment.
  • a method using an interaction with the target is generally used.
  • the method using the transistor can analyze a target having a charge, there is a problem that a target having little or no charge cannot be analyzed like a steroid skeleton-containing compound containing cortisol. It was.
  • the steroid skeleton-containing compound detection device of the present invention includes a transistor in which a nucleic acid sensor for detecting a steroid skeleton-containing compound is disposed,
  • the nucleic acid sensor is A stereogenic region (D) that forms a predetermined stereostructure and a binding region (A) that binds to the steroid skeleton-containing compound;
  • the stereogenic region (D) is inhibited from forming the stereostructure
  • the three-dimensional formation region (D) forms the three-dimensional structure.
  • the steroid skeleton-containing compound is present in a solution containing an electrolyte,
  • the number of nucleotide residues constituting the nucleic acid sensor in the Debye length range of the solution is increased or decreased as compared with the inhibition of formation of the
  • the method for detecting a steroid skeleton-containing compound of the present invention comprises a contact step of bringing a sample into contact with the detection device of the present invention, and an increase or decrease in the number of nucleotide residues constituting a nucleic acid sensor in the Debye length range of the detection device. By detecting a steroid skeleton-containing compound in the sample.
  • FIG. 1 is a schematic view showing a structural change of a nucleic acid sensor in the device of the present invention.
  • FIG. 2 is a schematic diagram showing the structural change of the nucleic acid sensor in the device of the present invention.
  • FIG. 3 is a graph showing ⁇ Vg when the single-stranded sensor in Example 1 of the present application is used.
  • FIG. 4 is a graph showing ⁇ Vg when the double-stranded sensor in Example 1 of the present application is used.
  • each region is also referred to as a nucleic acid region.
  • the single-stranded nucleic acid sensor described below can also be referred to as a single-stranded sensor, for example, and the double-stranded nucleic acid sensor can also be referred to as a double-stranded sensor, for example.
  • the switch-OFF or turn-OFF
  • the formation of the predetermined structure is indicated by a switch-ON (or turn- ON).
  • the three-dimensional formation region (D) may form the predetermined three-dimensional structure by contact with a compound.
  • the other three-dimensional structure is, for example, a three-dimensional structure different from the predetermined structure.
  • specific examples of the other three-dimensional structure for example, specific examples of the predetermined structure can be used.
  • the G-quartet (also referred to as “G-tetrad”) is generally known as a surface structure in which G (guanine) is a tetramer.
  • the G formation region (G) is, for example, a region having a plurality of bases G and forming a G-quartet structure with the plurality of bases G in the region.
  • the G-quartet structure may be, for example, a parallel type or an anti-parallel type, and is preferably a parallel type.
  • the number of G-quartet structures formed in the G formation region (G) is not particularly limited, and may be one surface or a plurality of two or more surfaces.
  • G preferably forms a guanine quadruplex (or G-quadruplex) structure in which multiple G-quartets are stacked.
  • the sequence of the G-forming region (G) may be any sequence that forms the G-quartet structure, and more preferably a sequence that forms a guanine quadruplex structure.
  • sequence of the G-forming region (G) for example, a sequence of a known nucleic acid molecule that forms the G-quartet structure can be used.
  • known nucleic acid molecule include nucleic acid molecules such as the following articles (1) to (4). (1) Travascio et al., Chem. Biol., 1998, vol.5, p.505-517 (2) Cheng et al., Biochemistry, 2009, vol.48, p.7817-7823 (3) Teller et al., Anal. Chem., 2009, vol.81, p.9114-9119 (4) Tao et al., Anal. Chem., 2009, vol.81, p.2144-2149
  • the sequence of the three-dimensional formation region (D) can be, for example, the sequence of a known nucleic acid molecule that forms the pseudoknot structure.
  • the known nucleic acid molecule include nucleic acid molecules such as the following paper (6). (6) Calliste Reiling et al., “Loop Contributions to the Folding Thermodynamics of DNA Straight Hairpin Loops and Pseudoknots”, 2015, J. Phys. Chem. B, vol.119, pp.1939-1946
  • the solid formation region (D) may be, for example, a single-stranded type or a double-stranded type.
  • the single-stranded type can form a predetermined structure in, for example, a single-stranded three-dimensional formation region (D), and the double-stranded type includes, for example, a first region (D1) and a second region (D2).
  • a predetermined structure can be formed between the first region (D1) and the second region (D2).
  • the latter double-stranded type includes, for example, a structure in which the first region and the second region are indirectly linked, and will be specifically described in the nucleic acid sensor (iv) described later.
  • the length of the single-stranded solid formation region (D) is not particularly limited, and the lower limit is, for example, 11 base length, 13 base length, 15 base length, and the upper limit is, for example, 60 base length, It is 36 bases long and 18 bases long.
  • the lengths of the first region (D1) and the second region (D2) are not particularly limited, and both may be the same or different.
  • the length of the first region (D1) is not particularly limited, and the lower limit is, for example, 7 base length, 8 base length, 10 base length, and the upper limit is, for example, 30 base length, 20 base length, 10
  • the base length is, for example, 7 to 30 bases, 7 to 20 bases, or 7 to 10 bases.
  • the length of the second region (D2) is not particularly limited, and the lower limit is, for example, 7 base length, 8 base length, 10 base length, and the upper limit is, for example, 30 base length, 20 base length, 10
  • the base length is, for example, 7 to 30 bases, 7 to 20 bases, or 7 to 10 bases.
  • the steroid compound means a compound having a steroid skeleton represented by the following formula (1).
  • the steroid skeleton-containing compound includes, for example, cortisol, cholesterol, progesterone, 11-deonecicorticosterone, corticosterone, aldosterone, cortisone, dehydroepiandrosterone, dehydroepiandrosterone represented by the following formula (2): Androsterone sulfate, dihydrotestosterone, androsterone, epiandrosterone, testosterone, 17 ⁇ -estradiol, estrone, estriol and the like.
  • the length of the binding region (A) is not particularly limited, and the lower limit is, for example, 12 base length, 15 base length, 18 base length, and the upper limit is, for example, 140 base length, 80 base length, 60 bases
  • the range is, for example, 12 to 140 bases long, 15 to 80 bases long, 18 to 60 bases long.
  • the phrase “the other sequence is complementary to a certain sequence” means, for example, a sequence that can be annealed between the two. The annealing is also referred to as stem formation, for example.
  • “complementary” means, for example, that complementarity when two kinds of sequences are aligned is, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more. Yes, preferably 100%, ie fully complementary.
  • the other sequence is complementary to a certain sequence when the sequence is directed from the 5 ′ side to the 3 ′ side, and the sequence is directed from the other 3 ′ side to the 5 ′ side. Means that the bases of each other are complementary.
  • the nucleic acid sensor (I) is, for example, a double-stranded nucleic acid sensor composed of a first strand (ss1) and a second strand (ss2),
  • the first strand (ss1) has the three-dimensional region (D) and the binding region (A) in this order
  • the second strand (ss2) has a stem forming region (S D ) and a stem forming region (S A ) in this order
  • the stem forming region (S D ) has a sequence complementary to the three-dimensional forming region (D)
  • the stem forming region (S A ) has a sequence complementary to the binding region (A)
  • the stereogenic region (D) is inhibited from forming the stereostructure and hybridizes with the second chain (ss2)
  • the three-dimensional formation region (D) forms the three-dimensional structure by contacting the steroid skeleton-
  • the three-dimensional formation region (D) is, for example, the single-stranded type.
  • the binding nucleic acid molecule such as an aptamer generally changes to a more stable structure by binding with the steroid compound and can bind to the steroid compound.
  • binding region such as an aptamer
  • the three-dimensional structure of a nucleic acid sequence such as a G-quartet structure is generally considered to have a higher abundance of relatively stable ones.
  • the sensor (I) in the absence of the steroid compound, has the three-dimensional formation region (D) and the second chain (ss2) of the first chain (ss1).
  • the binding region (A) of the first chain (ss1) and the stem formation region (S A ) of the second chain (ss2) are annealed, so that in the binding region (A), the steroid The formation of a more stable structure for binding to the compound is blocked, and the structure in a state not bonded to the steroid compound is maintained.
  • the sensor (I) can anneal the binding region (A) and the stem formation region (S A ) by contacting the steroid compound with the binding region (A).
  • the binding region (A) changes to the stable structure. Accordingly, the annealing of the three-dimensional formation region (D) and the stem formation region (S D ) is released, and the three-dimensional structure is formed in the region of the three-dimensional formation region (D) (switch-ON). . Also, as shown in FIG. 1B, annealing between the binding region (A) and the stem formation region (S A ), and between the three-dimensional formation region (D) and the stem formation region (S D ) When the annealing is released, the first strand (ss1) is dissociated from the second strand (ss2), and as a result, the first strand (ss1) can move out of the Debye length range. It becomes.
  • the steroid compound in the presence of the steroid compound, that is, when the three-dimensional structure is formed, the number of nucleotides of the Debye length is in the absence of the steroid compound, that is, the formation of the three-dimensional structure. Therefore, the steroid compound can be analyzed qualitatively or quantitatively.
  • the second chain (ss2) is described as being disposed in the transistor. However, as will be described later, the first chain (ss1) is disposed in the transistor. Also good.
  • the ss1 includes the first chain (ss1) and the second chain (ss2), and in the presence of the steroid compound, the first chain (ss1) or the second chain (ss2). ) Dissociates and moves, for example, outside the Debye length range. For this reason, even when the steroid compound has a charge, in the presence of the steroid compound, the charge in the Debye length range is equal to the number of dissociated first chains (ss1) or second chains (ss2). Fluctuate accordingly. For this reason, the sensor (I) is excellent in versatility because, for example, the influence of the charge of the steroid compound is reduced.
  • the three-dimensional formation region (D) and the stem formation region (S D ) are annealed in the order of the regions, and the binding region (A) and the stem formation region (S A ) are annealed. And the order of annealing.
  • the following order can be illustrated as a specific example. (1) ss1 5'- AD-3 ' ss2 3'- S A -S D -5 ' (2) ss1 5'- DA-3 ' ss2 3'- S D -S A -5 '
  • the sensor (I) may be connected, for example, directly or indirectly between the regions.
  • the direct connection means that, for example, the 3 ′ end of one region and the 5 ′ end of the other region are directly bonded, and the indirect connection is, for example, 3 of one region. It means that the “end” and the 5 ′ end of the other region are bound via an intervening linker region.
  • the intervening linker region may be, for example, a nucleic acid sequence or a non-nucleic acid sequence, preferably the former.
  • the sensor (I) includes, for example, the stem formation region (A) between the binding region (A) and the three-dimensional formation region (D) in the first strand (ss1) and the second strand (ss2). It is preferable to have the intervening linker region between S D ) and the stem formation region (S A ).
  • the intervening linker region (L 1 ) in the first strand (ss1) and the intervening linker region (L 2 ) in the second strand (ss2) are preferably non-complementary sequences.
  • the formation of a three-dimensional structure is turned on and off as follows.
  • the binding region (A) and the stem formation region (S A ) the three-dimensional formation region (D) and the stem formation region (S D ) each form a stem,
  • the intervening linker region (L 1 ) and the intervening linker region (L 2 ) form an internal loop to inhibit the formation of the three-dimensional structure of the three-dimensional formation region (D).
  • the formation of each stem is released by the contact of the steroid compound with the binding region (A), and the three-dimensional structure is formed in the three-dimensional formation region (D).
  • the lengths of the stem formation region (S A ) and the stem formation region (S D ) are not particularly limited.
  • the length of the stem formation region (S A ) is, for example, 1 to 60 bases long, 1 to 10 bases long, or 1 to 7 bases long.
  • the length of the stem formation region (S D ) is, for example, 1 to 30 bases, 0 to 10 bases, 1 to 10 bases, 0 to 7 bases, or 1 to 7 bases.
  • the stem formation region (S A ) and the stem formation region (S D ) may have the same length, the former may be long, or the latter may be long.
  • the lengths of the intervening linker regions (L 1 ) and (L 2 ) are not particularly limited.
  • the lengths of the intervening linker regions (L 1 ) and (L 2 ) are, for example, 0 to 30 bases, 1 to 30 bases, 1 to 15 bases, and 1 to 6 bases, respectively.
  • the lengths of the intervening linker regions (L 1 ) and (L 2 ) may be the same or different, for example. In the latter case, the difference in length between the intervening linker regions (L 1 ) and (L 2 ) is not particularly limited, and is, for example, 1 to 10 bases long, 1 or 2 bases long, and 1 base long.
  • the lengths of the first strand (ss1) and the second strand (ss2) are not particularly limited.
  • the length of the first strand (ss1) is, for example, 40 to 200 bases long, 42 to 100 bases long, 45 to 60 bases long.
  • the length of the second strand (ss2) is, for example, 4 to 120 bases long, 5 to 25 bases long, or 10 to 15 bases long.
  • the first chain (ss1) and the second chain (ss2) may be directly or indirectly linked.
  • the sensor (I) can be referred to as a single-stranded nucleic acid sensor, for example, and the first strand (ss1)
  • the second strand (ss2) can be referred to as a first region and a second region, respectively.
  • the direct connection means that, for example, the 3 ′ end of one region and the 5 ′ end of the other region are directly bonded, and the indirect connection is, for example, 3 of one region.
  • the intervening linker region may be, for example, a nucleic acid sequence or a non-nucleic acid sequence, preferably the former.
  • the length of the intervening linker region is not particularly limited and is, for example, 1 to 60 bases long.
  • the first region, the intervening linker region, and the second region may be connected in this order from the 5 ′ side. They may be connected in this order from the 3 ′ side, preferably the former.
  • one end of the first chain (ss1) or the second chain (ss2) may be connected to the transistor.
  • a linker region may be further added to the one end or both ends of the first strand (ss1) and the second strand (ss2).
  • the linker region added to the terminal is also referred to as an additional linker region.
  • the length of the additional linker region is not particularly limited and is, for example, 1 to 60 bases long.
  • one end of the first strand (ss1) or the second strand (ss2) may be connected to the transistor via an additional linker region.
  • (A) a polynucleotide comprising the base sequence of (a1), (a2) or (a3) below (a1) SEQ ID NO: 1 (a2) in the base sequence of SEQ ID NO: 1, wherein one or several bases are It consists of a base sequence deleted, substituted, inserted and / or added, the region corresponding to the binding region (A) binds to cortisol, and the region corresponding to the stereogenic region (D) is the predetermined three-dimensional structure
  • the polynucleotide (a3) comprising the nucleotide sequence having the identity of 80% or more with respect to the nucleotide sequence of SEQ ID NO: 1, the region corresponding to the binding region (A) binds to cortisol, and the three-dimensional formation
  • (B) a polynucleotide consisting of the base sequence of (b1), (b2), or (b3) below (b1) SEQ ID NO: 2 (b2) in the base sequence of SEQ ID NO: 2, wherein one or several bases deleted, substituted, inserted and / or added in the nucleotide sequence, wherein the system forming area (S a) a region corresponding to the said bound to the binding region (a) of the polynucleotide of (a) (annealing) A region corresponding to the stem-forming region (S D ) with respect to the base sequence of the polynucleotide (b3) SEQ ID NO: 2 that binds (anneals) to the three-dimensional formation region (D) of the polynucleotide of (a), the base sequence having 80% or more identity, coupled to the coupling region of a polynucleotide of the stem forming regions (S a) a region corresponding to said (a) (a) Annealing), and said
  • the polynucleotide (a1) is a polynucleotide having the base sequence of SEQ ID NO: 1 below.
  • the base sequence indicated by the underline at the 5 ′ end corresponds to the three-dimensional formation region (D)
  • the base sequence indicated by the underline enclosed in parentheses at the 3 ′ end is the binding
  • the base sequence corresponding to the region (A) and not underlined corresponds to the intervening linker region (L 1 ).
  • First strand (ss1) (SEQ ID NO: 1): 5'- GGGTGGGAGGGTCGGG TTT [ GCAAGTTCTTCGCTCGTACTTGC ] -3 '
  • “one or several” means, for example, that the polynucleotide of (a2) binds to cortisol in the region corresponding to the binding region (A), and the three-dimensional region (D
  • the region corresponding to) may be a range that forms the predetermined three-dimensional structure.
  • the “1 or several” is, for example, 1 to 8, 1 to 4, 1 or 2, or 1 in the base sequence of (a1).
  • the numerical range of numbers such as the number of bases and the number of sequences, for example, discloses all positive integers belonging to the range. That is, for example, the description “1 to 5 bases” means all disclosures of “1, 2, 3, 4, 5 bases” (the same applies hereinafter).
  • identity means, for example, that the polynucleotide of (a3) binds to cortisol in the region corresponding to the binding region (A), and the stereogenic region (D)
  • the corresponding region may be a range that forms the predetermined three-dimensional structure.
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the identity can be calculated with default parameters using analysis software such as BLAST and FASTA (hereinafter the same).
  • the polynucleotide (b1) is a polynucleotide having the base sequence of SEQ ID NO: 2 below.
  • the base sequence indicated by the underline at the 5 ′ end corresponds to the stem formation region (S A )
  • the base sequence indicated by the underline enclosed in parentheses at the 3 ′ end is The base sequence without underline corresponding to the stem formation region (S D ) corresponds to the intervening linker region (L 2 ).
  • Second strand (ss2) (SEQ ID NO: 2): 5'- AACTTGC TTT [ CCCGACCC ] -3 '
  • the polynucleotide of (b2) “one or several” means, for example, that the polynucleotide of (b2) is a region corresponding to the stem forming region (S A ) of the polynucleotide of (a).
  • the region that binds to the binding region (A) and corresponds to the stem forming region (S D ) may be in a range that binds to the three-dimensional forming region (D) of the polynucleotide of (a).
  • the polynucleotide (b2) may be any of the polynucleotides (a), the stem formation region (S A ), and the stem formation region (S D ) as long as they can be combined as described above.
  • the “1 or several” is, for example, 1 to 4, 1 to 3, 1 or 2, or 1 in the base sequence of (b1).
  • identity means, for example, that the polynucleotide of (b3) is a region corresponding to the stem-forming region (S A ), and the binding region of the polynucleotide of (a)
  • the region that binds to (A) and corresponds to the stem forming region (S D ) may be in a range that binds to the three-dimensional forming region (D) of the polynucleotide of (a).
  • the polynucleotide (b3) may be any one of the polynucleotides (a), the stem formation region (S A ), and the stem formation region (S D ) as long as they can be combined as described above.
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the binding region (A) in the absence of the steroid compound, the binding region (A) does not form a more stable structure for binding to the steroid compound, and the stem-forming region (S A )
  • the formation of the stable structure is blocked in the binding region (A), and the structure that is not bound to the steroid compound is maintained. Accordingly, the formation of the three-dimensional structure of the three-dimensional formation region (D) is inhibited (switch-OFF), and the stem formation region (S D ) anneals to the three-dimensional formation region (D). .
  • the sensor (I) in the absence of the steroid compound, the first strand (ss1) and the second strand (ss2) are hybridized.
  • the sensor (I) changes the binding region (A) into the stable structure by the contact of the steroid compound with the binding region (A), thereby forming the stem formation.
  • the region (S A ) does not anneal to the binding region (A).
  • the stem formation region (S D ) does not anneal to the solid formation region (D), and the solid structure is formed in the region of the solid formation region (D) (switch -ON).
  • the annealing between the bonding region (A) and the stem formation region (S A ) and the annealing between the three-dimensional formation region (D) and the stem formation region (S D ) are not formed, so that the first The strand (ss1) does not hybridize to the second strand (ss2), and the first strand (ss1) can move out of the Debye length range. Therefore, according to the sensor (I), in the presence of the steroid compound, that is, when the three-dimensional structure is formed, the number of nucleotides of the Debye length is in the absence of the steroid compound, that is, the formation of the three-dimensional structure. Therefore, the steroid compound can be analyzed qualitatively or quantitatively.
  • the second chain (ss2) has been described as an example arranged in the transistor, the first chain (ss1) may be arranged in the transistor.
  • the sensor (II) can be turned on and off by the three-dimensional structure of the three-dimensional formation region (D) depending on the presence or absence of the steroid compound based on the following mechanism, for example. It is presumed that the number of nucleotide residues constituting the sensor increases in the range of the Debye length. Note that the present invention is not limited to this mechanism. As shown in FIG. 2A, in the sensor (II), in the absence of the steroid compound, formation of the three-dimensional structure of the three-dimensional formation region (D) is inhibited in the molecule (switch-OFF ).
  • the sensor (II) is more stable for the binding region (A) to bind to the steroid compound by the contact of the steroid compound with the binding region (A). Change to structure. Along with this, the three-dimensional structure is formed in the region of the three-dimensional formation region (D) (switch-ON). Further, as shown in FIG. 2B, the binding region (A) changes to the stable three-dimensional structure, and the three-dimensional formation region (D) forms a three-dimensional structure, whereby the sensor ( II) shrinks to the transistor side, for example.
  • the steroid compound in the presence of the steroid compound, that is, when the three-dimensional structure is formed, the number of nucleotides of the Debye length is in the absence of the steroid compound, that is, the formation of the three-dimensional structure. Therefore, the steroid compound can be analyzed qualitatively or quantitatively.
  • the senor (II) is, for example, at least one sensor selected from the group consisting of the following (i) to (iv) and (v).
  • the sensor (II) may include, for example, one type of sensor or may include two or more types of sensors.
  • Nucleic acid sensor (i) The sensor (i) has, for example, the three-dimensional formation region (D), the blocking region (B), and the binding region (A) in this order,
  • the blocking region (B) is complementary to a partial region (Dp) in the three-dimensional region (D);
  • a terminal region (Ab) on the blocking region (B) side in the binding region (A) is complementary to a region (Df) adjacent to the partial region (Dp) in the three-dimensional formation region (D), and
  • the single-stranded nucleic acid sensor is complementary to a terminal region (Af) opposite to the blocking region (B).
  • stem formation between the partial region (Dp) of the stereogenic region (D) and the blocking region (B) and the adjacent region (Df) of the stereogenic region (D) And stem formation of the terminal region (Ab) of the binding region (A) occurs.
  • the former stem formation inhibits the formation of the three-dimensional structure of the three-dimensional formation region (D) (switch-OFF), and the latter stem formation causes the binding region (A) to bind to the steroid compound.
  • the formation of a more stable structure is blocked, and the structure that is not bonded to the steroid compound is maintained.
  • the binding region (A) changes to the stable structure by the contact of the steroid compound with the binding region (A).
  • stem formation in the binding region (A) is released, and the steroid compound binds to the binding region (A) changed to the stable structure.
  • the stem formation of the three-dimensional formation region (D) is also released, and the three-dimensional formation region (D) becomes more
  • the structure changes to a stable structure, and as a result, a three-dimensional structure is formed in the region of the three-dimensional formation region (D) (switch-ON).
  • the sensor (i) may further have a stabilization region (S).
  • the conversion regions (S) are preferably connected in this order.
  • the stabilization region (S) is optional and may not be included.
  • the stabilization region (S) is, for example, a sequence for stabilizing the structure when the binding region (A) binds to the steroid compound.
  • the stabilization region (S) is, for example, complementary to the blocking region (B) or complementary to a part thereof, specifically, on the binding region (A) side in the blocking region (B). It is preferably complementary to the terminal region (Ba).
  • the stabilization region (S) connected to the binding region (A) and the binding region ( A stem is also formed between the blocking region (B) and the terminal region (Ba) connected to A).
  • the order of the three-dimensional formation region (D), the blocking region (B), the binding region (A), and the optional stabilization region (S) is not particularly limited. They may be connected in this order from the 5 ′ side, or may be connected in this order from the 3 ′ side, preferably the former.
  • the arrangement and length of the blocking region (B) are not particularly limited, and can be appropriately set according to, for example, the arrangement and length of the three-dimensional formation region (D).
  • the length of the blocking region (B) is not particularly limited, and the lower limit is, for example, 1 base length, 2 base lengths, 3 base lengths, and the upper limit is, for example, 20 base lengths, 15 base lengths, 10 bases
  • the range is, for example, 1 to 20 bases long, 2 to 15 bases long, and 3 to 10 bases long.
  • the position of the partial region (Dp) in the solid formation region (D), that is, the annealing region of the blocking region (B) in the solid formation region (D) is not particularly limited.
  • the partial region (Dp) Can be set under the following conditions, for example.
  • the three-dimensional formation region (D) is a region adjacent to the partial region (Dp), which is the blocking region (B) side end of the partial region (Dp) and the three-dimensional formation region (D) in the blocking region (B).
  • the lower limit of the length of the region (Db) between the side ends is, for example, 3 base length, 4 base length, 5 base length
  • the upper limit is, for example, 40 base length, 30 base length, 20 base length.
  • the range is, for example, 3 to 40 bases long, 4 to 30 bases long, and 5 to 20 bases long.
  • the lower limit of the length of the region (Df) adjacent to the partial region (Dp) in the three-dimensional region (D) and opposite to the blocking region (B) side is, for example, 0 base length
  • the upper limit is, for example, 40 base length, 30 base length, 20 base length
  • the range is, for example, 0-40 base length, 1-30 base length, It is 20 bases long.
  • the length of the terminal region (Ab) in the binding region (A) complementary to the adjacent region (Df) of the three-dimensional region (D) is not particularly limited, and the lower limit is, for example, one base length
  • the upper limit is, for example, 20 base length, 8 base length, 3 base length, and the range is, for example, 1-20 base length, 1-8 base length, 1-3 base length.
  • the sequence and length of the stabilization region (S) are not particularly limited, and are appropriately determined according to, for example, the sequence and length of the blocking region (B), the sequence and length of the binding region (A), and the like. it can.
  • the lower limit of the length of the stabilization region (S) is, for example, 0 base length and 1 base length
  • the upper limit is, for example, 10 base length, 5 base length, 3 base length
  • the range is For example, the length is 0 to 10 bases, 1 to 5 bases, or 1 to 3 bases.
  • the stabilization region (S) is complementary to the entire blocking region (B)
  • the blocking region (B) has the same length as the stabilization region (S).
  • the stabilization region (S) is complementary to a part of the blocking region (B), a part of the blocking region (B), for example, the terminal region (Ba) It is the same length as (S).
  • the total length of the sensor (i) is not particularly limited, and the lower limit is, for example, 25 base length, 35 base length, 40 base length, and the upper limit is, for example, 200 base length, 120 base length, 80 The base length is, for example, 25 to 200 bases, 35 to 120 bases, 40 to 80 bases.
  • one end of the sensor (i) may be connected to the transistor.
  • the additional linker region may be further added to one end or both ends.
  • the length of the additional linker region is not particularly limited, and for example, the above description can be used.
  • one end of the sensor (i) may be connected to the transistor via the additional linker region.
  • Nucleic acid sensor (ii) The sensor (ii) has, for example, the three-dimensional formation region (D), the blocking region (B), the binding region (A), and the stabilization region (S) in this order,
  • the blocking region (B) is complementary to a partial region (Dp) of the three-dimensional formation region (D);
  • the terminal region (Ba) on the binding region (A) side of the blocking region (B) is a single-stranded nucleic acid sensor that is complementary to the stabilization region (S).
  • the three-dimensional formation region (D) is, for example, the single-stranded type.
  • the binding region (A) is preferably a sequence that alone does not form intramolecular annealing necessary for binding to the steroid compound. Then, the sensor (ii) is obtained by annealing the terminal region (Ba) of the blocking region (B) adjacent to the binding region (A) and the stabilization region (S) in the presence of the steroid compound. It is preferable that a stable structure for binding to the steroid compound is formed from the whole of the binding region (A), the terminal region (Ba), and the stabilization region (S).
  • the formation of the three-dimensional structure of the three-dimensional formation region (D) is controlled to ON-OFF depending on the presence or absence of the steroid compound.
  • the number of nucleotide residues constituting the sensor is estimated to increase. Note that the present invention is not limited to this mechanism.
  • the partial region (Dp) of the three-dimensional formation region (D) is complementary to the blocking region (B)
  • stem formation is possible in this complementary relationship. For this reason, in the absence of the steroid compound, stem formation occurs between the partial region (Dp) of the stereogenic region (D) and the blocking region (B).
  • the binding region (A) is a sequence that does not form intramolecular annealing necessary for binding to the steroid compound by itself, formation of a more stable structure for binding to the steroid compound is blocked. The structure in a state where it is not bonded to the steroid compound is maintained. On the other hand, in the presence of the steroid compound, the binding region (A) changes to the stable structure by the contact of the steroid compound with the binding region (A).
  • the stem formation of the blocking region (B) and the partial region (Dp) of the three-dimensional formation region (D) is released, and the terminal region (Ba) of the blocking region (B) and the stable region are newly added.
  • the stem is formed by annealing with the activating region (S), and this stem plays a role of intramolecular annealing necessary for the binding region (A) to bind to the steroid compound, and the stem and the binding region
  • the stable structure is formed from the entirety of (A), and the steroid compound is bound to the binding region (A).
  • the order of the three-dimensional formation region (D), the blocking region (B), the binding region (A), and the stabilization region (S) is not particularly limited.
  • 5 ′ They may be connected in this order from the side, or may be connected in this order from the 3 ′ side, preferably the former.
  • the description of the sensor (i) can be used unless otherwise indicated.
  • the three-dimensional formation region (D), the blocking region (B), and the stabilization region (S) are the same as, for example, the sensor (i).
  • the length of the terminal region (Ba) complementary to the stabilization region (S) is not particularly limited, and the lower limit is, for example, one base length, and the upper limit is, for example, 15 base length, 10 base length and 3 base length, and the range is, for example, 1 to 10 base length, 1 to 5 base length, and 1 to 3 base length.
  • the total length of the sensor (ii) is not particularly limited, and the lower limit is, for example, 25 base length, 35 base length, 40 base length, and the upper limit is, for example, 200 base length, 120 base length, 80 The base length is, for example, 25 to 200 bases, 35 to 120 bases, 40 to 80 bases.
  • the sensor (iii) includes, for example, the three-dimensional formation region (D), the stem formation region (S D ), the binding region (A), and the stem formation region (S A ).
  • the stem forming region (S D ) has a sequence complementary to the three-dimensional forming region (D)
  • the stem forming region (S A ) is a single-stranded nucleic acid sensor having a sequence complementary to the binding region (A).
  • the three-dimensional formation region (D) is, for example, the single-stranded type.
  • the binding region (A) and the stem formation region (S A ) are annealed, so that the binding region (A) has a more stable structure for binding to the steroid compound. Formation is blocked and the structure is not bonded to the steroid compound.
  • the sensor (iii) causes the annealing of the binding region (A) and the stem formation region (S A ) by the contact of the steroid compound with the binding region (A). When released, the structure of the binding region (A) changes to the stable structure.
  • the annealing of the three-dimensional formation region (D) and the stem formation region (S D ) is released, and the three-dimensional structure is formed in the region of the three-dimensional formation region (D) (switch-ON).
  • the sensor (iii) is, for example, on the transistor side. Shrink. Therefore, according to the sensor (iii), in the presence of the steroid compound, that is, when the three-dimensional structure is formed, the number of nucleotides of the Debye length is in the absence of the steroid compound, that is, the formation of the three-dimensional structure. Therefore, the steroid compound can be analyzed qualitatively or quantitatively.
  • the three-dimensional formation region (D) and the stem formation region (S D ) are annealed in the molecule, and the binding region (A) and the stem formation are performed in the sensor (iii).
  • the order of annealing with the region (S A ) is sufficient.
  • the following order can be illustrated as a specific example. (1) 5'- A-S D -D-S A -3 ' (2) 5'-S A -DSD D -A -3 ' (3) 5'- D-S A -A-S D -3 ' (4) 5'-S D -AS A -D -3 '
  • the formation of a three-dimensional structure is turned on and off as follows.
  • the binding region (A) and the stem formation region (S A ), the three-dimensional formation region (D) and the stem formation region (S D ) each form a stem, and the three-dimensional formation
  • the formation of the three-dimensional structure in the region (D) is inhibited.
  • the formation of the respective stems is released by the contact of the steroid compound with the binding region (A), and the three-dimensional structure is formed in the three-dimensional formation region (D).
  • the stem forming region (S D ) is complementary to the 3′-side region of the three-dimensional forming region (D), and the stem forming region (S A ) It is preferably complementary to the 3 ′ region of the region (A).
  • the stem formation region (S D ) is complementary to the 5′-side region of the three-dimensional formation region (D), and the stem formation region (S A ) It is preferably complementary to the 5 ′ region of the region (A).
  • the sensor (iii) preferably has, for example, two intervening linker regions that are non-complementary to each other as the intervening linker region.
  • the positions of the two intervening linker regions are not particularly limited.
  • the following order can be exemplified for the forms (1) to (4) further having two intervening linker regions.
  • the intervening linker region linked to the binding region (A) is indicated by (L 1 )
  • the intervening linker region linked to the stereogenic region (D) is indicated by (L 2 ).
  • the sensor (iii) may have, for example, both (L 1 ) and (L 2 ) as an intervening linker region, or may have only one of them.
  • the formation of the three-dimensional structure is turned on and off as follows.
  • the binding region (A) and the stem formation region (S A ) the three-dimensional formation region (D) and the stem formation region (S D ) each form a stem
  • the intervening linker region (L 1 ) and the intervening linker region (L 2 ) form an internal loop to inhibit the formation of the three-dimensional structure of the three-dimensional formation region (D).
  • the formation of the respective stems is released by the contact of the steroid compound with the binding region (A), and a three-dimensional structure is formed in the three-dimensional formation region (D).
  • the lengths of the intervening linker regions (L 1 ) and (L 2 ) are not particularly limited.
  • the lengths of the intervening linker regions (L 1 ) and (L 2 ) are, for example, 0 to 30 bases, 1 to 30 bases, 1 to 15 bases, and 1 to 6 bases, respectively.
  • the lengths of the intervening linker regions (L 1 ) and (L 2 ) may be the same or different, for example. In the latter case, the difference in length between the intervening linker regions (L 1 ) and (L 2 ) is not particularly limited, and is, for example, 1 to 10 bases long, 1 or 2 bases long, and 1 base long.
  • the length of the sensor (iii) is not particularly limited.
  • the length of the sensor (iii) is, for example, 40 to 120 bases long, 45 to 100 bases long, 50 to 80 bases long.
  • one end of the sensor (iii) may be connected to the transistor.
  • the senor (iii) includes, for example, the following polynucleotide (c).
  • (C) polynucleotide (c2) comprising the nucleotide sequence of SEQ ID NO: 3 (c1), polynucleotide (c1), (c3) or (c3) below:
  • (c1) In the nucleotide sequence of SEQ ID NO: 3, one or several bases It consists of a base sequence deleted, substituted, inserted and / or added, the region corresponding to the binding region (A) binds to cortisol, and the region corresponding to the stereogenic region (D) is the predetermined three-dimensional structure
  • the region corresponding to the stem formation region (S A ) is bonded (annealed) to the binding region (A), and the region corresponding to the stem formation region (S D ) is the solid formation region (D).
  • the polynucleotide (c3) that binds (anneals) to the nucleotide sequence of SEQ ID NO: 3 consists of a nucleotide sequence having at least 80% identity, and the region corresponding to the binding region (A) A region that binds to lutisol and that corresponds to the three-dimensional region (D) forms the predetermined three-dimensional structure, and a region that corresponds to the stem-forming region (S A ) binds to the binding region (A) (annealing). polynucleotide, and said stem forming regions (S D) corresponding to the region is bound (annealing) on the three-dimensional formation region (D)
  • the polynucleotide (c1) is a polynucleotide having the base sequence of SEQ ID NO: 3 below.
  • the base sequence indicated by the underline enclosed in parentheses is the stem-forming region (S A ) and the intervening linker region from the 5 ′ end to the 3 ′ end, respectively ( L 2 ), the three-dimensional formation region (D), the stem formation region (S D ), the intervening linker region corresponds to (L 1 ), and the binding region (A).
  • Single-stranded nucleic acid sensor (SEQ ID NO: 3): 5 '-[ ACCTCTG ] [ T ] [ GGGTGGGAGGGTCGGG ] [ CCC ] [ T ] [ CAGAGGTCTCTTTGCCCGTGAACTCTG ] -3'
  • identity means, for example, that the polynucleotide of (c3) binds to cortisol in the region corresponding to the binding region (A), and the stereogenic region (D)
  • the corresponding region forms the predetermined three-dimensional structure, the region corresponding to the stem forming region (S A ) is bonded to the bonding region (A), and the region corresponding to the stem forming region (S D ) is the What is necessary is just the range couple
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the additional linker region may be further added to one end or both ends.
  • the length of the additional linker region is not particularly limited, and for example, the above description can be used.
  • one end of the sensor (iii) may be connected to the transistor via the additional linker region.
  • Nucleic acid sensor (iv) The sensor (iv) has, for example, the three-dimensional formation region (D) and the binding region (A),
  • the three-dimensional formation region (D) includes a first region (D1) and a second region (D2), and forms a three-dimensional structure with the first region (D1) and the second region (D2).
  • a single-stranded nucleic acid sensor having the first region (D1) on one end side of the binding region (A) and the second region (D2) on the other end side of the binding region (A) It is.
  • the three-dimensional formation region (D) is, for example, the double-stranded type (hereinafter also referred to as “split type”).
  • the split-type three-dimensional formation region (D) is a molecule that includes the first region (D1) and the second region (D2), and the pair forms a three-dimensional structure.
  • the first region (D1) and the second region (D2) may each be a sequence that forms the three-dimensional structure, and more preferably a guanine quadruplex structure. Is an array.
  • the formation of the three-dimensional structure of the three-dimensional formation region (D) is controlled to be ON-OFF depending on the presence or absence of the steroid compound.
  • the number of nucleotide residues constituting the sensor is estimated to increase. Note that the present invention is not limited to this mechanism.
  • the sensor (iv) includes a pair of the first region (D1) and the second region (D2) that form a three-dimensional structure via the coupling region (A). Are located apart. Thus, since the first region (D1) and the second region (D2) are arranged at a distance, in the absence of the steroid compound, the first region (D1) and the second region (D2).
  • the formation of a three-dimensional structure is inhibited between the region (D2) (switch-OFF).
  • the structure of the binding region (A) in the presence of the steroid compound, has a stem-loop structure due to the contact of the steroid compound with the binding region (A). Changes to a more stable structure for bonding with. With the structural change of the coupling region (A), the first region (D1) and the second region (D2) approach each other, and the first region (D1) and the second region (D2) A three-dimensional structure is formed between them (switch-ON).
  • the bonding region (A) changes to the stable structure, and the three-dimensional formation region (D) forms the three-dimensional structure, so that the sensor (iv) is, for example, on the transistor side. Shrink. Therefore, according to the sensor (iv), in the presence of the steroid compound, that is, when the three-dimensional structure is formed, the number of nucleotides of the Debye length is in the absence of the steroid compound, that is, the formation of the three-dimensional structure. Therefore, the steroid compound can be analyzed qualitatively or quantitatively.
  • the first region (D1) and the second region (D2) may be arranged via the coupling region (A), and any one of the five of the coupling regions (A). It may be arranged on the 'side or 3' side.
  • the first region (D1) is disposed on the 5 ′ side of the coupling region (A)
  • the second region (D2) is disposed on the 3 ′ side of the coupling region (A). An example is shown.
  • the sensor (iv) includes the intervening linker region (first linker region (L 1 )) between the first region (D1) and the binding region (A). It is preferable that the intervening linker region (second linker region (L 2 )) is provided between two regions (D2) and the binding region (A).
  • the first linker region (L 1 ) and the second linker region (L 2 ) may be either one or preferably both. When both the first linker region (L 1 ) and the second linker region (L 2 ) are included, the respective lengths may be the same or different.
  • the length of the linker region is not particularly limited, and the lower limit is, for example, 1, 3, 5, 7, 9 bases, and the upper limit is, for example, 20, 15, 10 bases.
  • the distance between the first region (D1) and the second region (D2) can be sufficiently maintained. For this reason, for example, in the absence of the steroid compound, the formation of the three-dimensional structure by the first region (D1) and the second region (D2) is sufficiently suppressed, and in the absence of the steroid compound, The background based on the formation of the three-dimensional structure can be sufficiently reduced.
  • the sensor (iv) includes, for example, a sequence in which the first region (D1) and the second region (D2) are complementary to each other at the end opposite to the position of the binding region (A). It is preferable to have. Specifically, for example, when the first region (D1) is disposed on the 5 ′ side of the coupling region (A), the first region (D1) and the second region (D2) are: It is preferable that the 5 ′ end of the first region (D1) and the 3 ′ end of the second region (D2) have sequences complementary to each other. For example, when the first region (D1) is disposed on the 3 ′ side of the coupling region (A), the first region (D1) and the second region (D2) are the first region (D1).
  • the 3 ′ end of the region (D1) and the 5 ′ end of the second region (D2) preferably have complementary sequences.
  • a stem structure can be formed between the sequences by intramolecular annealing. It becomes possible. Therefore, for example, in the presence of the steroid compound, when the first region (D1) and the second region (D2) approach each other due to the structural change of the binding region (A) due to contact with the steroid compound, Formation of a stem structure between the arrays facilitates formation of a three-dimensional structure of the first region (D1) and the second region (D2).
  • (N) n1 and (N) m1 satisfy the following condition (1): N) n2 and (N) m2 preferably satisfy the following condition (2), and (N) n3 and (N) m3 preferably satisfy the following condition (3).
  • Condition (1) In (N) n1 and (N) m1 , the base sequence from the 5 ′ side of (N) n1 and the base sequence from the 3 ′ side of (N) m1 are complementary to each other, and n1 and m1 are The same 0 or a positive integer.
  • Condition (2) In (N) n2 and (N) m2 , the base sequence from the 5 ′ side of (N) n2 and the base sequence from the 3 ′ side of (N) m2 are non-complementary to each other, and n2 and m2 are Are positive integers, which may be the same or different.
  • N and (N) m3 are those in which n3 and m3 are 3 or 4, respectively, and may be the same or different, have three bases G, and when n3 or m3 is 4, (N) In n3 and (N) m3 , the second or third base is a base H other than G.
  • the condition (1) is a condition of (N) n1 at the 5 ′ end and (N) m1 at the 3 ′ end when the first region (D1) and the second region (D2) are aligned. .
  • the base sequence from the 5 ′ side of the (N) n1 and the base sequence from the 3 ′ side of the (N) m1 are complementary to each other and have the same length. Since (N) n1 and (N) m1 are complementary sequences of the same length, they can be said to be stem regions that form stems in an aligned state.
  • N1 and m1 may be the same 0 or a positive integer, and are, for example, 0, 1 to 10, and preferably 1, 2, or 3, respectively.
  • the condition (2) is a condition of (N) n2 and (N) m2 when the first region (D1) and the second region (D2) are aligned.
  • the base sequence of (N) n2 and the base sequence of (N) m2 are non-complementary to each other, and n2 and m2 may have the same length or different lengths. Since (N) n2 and (N) m2 are non-complementary sequences, they can be said to be regions that form an inner loop in an aligned state.
  • Examples of the base H that is a base other than G include A, C, T, and U, and preferably A, C, or T.
  • condition (3) include the following conditions (3-1), (3-2), and (3-3).
  • Condition (3-1) Among (N) n3 and (N) m3 , the sequence from one 5 ′ side is GHGG, and the sequence from the other 5 ′ side is GGG.
  • Condition (3-2) Among (N) n3 and (N) m3 , the sequence from one 5 ′ side is GGHG, and the sequence from the other 5 ′ side is GGG.
  • Condition (3-3) Both (N) n3 and (N) m3 sequences are GGG.
  • the length of the first region (D1) is not particularly limited, and the lower limit is, for example, 7 base length, 8 base length, 10 base length, and the upper limit is, for example, 30 base length, 20 base length, 10
  • the base length is, for example, 7 to 30 bases, 7 to 20 bases, or 7 to 10 bases.
  • the length of the second region (D2) is not particularly limited, and the lower limit thereof is, for example, 7 base length, 8 base length, 10 base length, and the upper limit thereof is, for example, 30 base length, 20 base length.
  • the range is, for example, 7 to 30 bases, 7 to 20 bases, or 7 to 10 bases.
  • the lengths of the first region (D1) and the second region (D2) may be the same or different.
  • one end of the sensor (iv) may be connected to the transistor.
  • Nucleic acid sensor (v) The sensor (v) has the three-dimensional formation region (D) and the binding region (A) in this order, The three-dimensional formation region (D) and the binding region (A) are single-stranded nucleic acid sensors having sequences complementary to each other.
  • the three-dimensional formation region (D) is, for example, the single-stranded type.
  • the binding region (A) and the three-dimensional formation region (D) are annealed in the molecule, so that the binding region (A) forms a more stable structure for binding to the steroid compound. Is blocked, and the structure in a state where it is not bonded to the steroid compound is maintained.
  • the structure of the binding region (A) changes to the stable structure by the contact of the steroid compound with the binding region (A).
  • the annealing in the region between the three-dimensional formation region (D) and the bonding region (A) is canceled, and the three-dimensional structure is formed in the region of the three-dimensional formation region (D) (switch-ON).
  • the coupling region (A) changes to the stable structure, and the three-dimensional formation region (D) forms the three-dimensional structure, so that the sensor (v) is, for example, on the transistor side. Shrink. Therefore, according to the sensor (v), in the presence of the steroid compound, that is, when the three-dimensional structure is formed, the number of nucleotides of the Debye length is in the absence of the steroid compound, that is, the formation of the three-dimensional structure. Therefore, the steroid compound can be analyzed qualitatively or quantitatively.
  • the three-dimensional formation region (D) and the binding region (A) are an arrangement from the 5 ′ side of the three-dimensional formation region (D) and a 3 ′ side of the binding region (A). Preferably have sequences complementary to each other.
  • the complementary sequence in the stereogenic region (D) and the complementary sequence in the binding region (A) can also be referred to as stem-forming regions (St), respectively, and the complementary sequence in the former stereogenic region (D) is
  • the stem formation region (S A ) for the binding region (A) and the complementary sequence in the latter binding region (A) can also be referred to as the stem formation region (S D ) for the three-dimensional formation region (D).
  • a part of the three-dimensional formation region (D) is the complementary sequence, that is, the stem formation region (S A ), and a part of the binding region (A) is, for example, the complementary sequence. That is, it is preferably the stem formation region (S D ).
  • the position of the complementary sequence in the three-dimensional region (D) and the position of the complementary sequence in the binding region (A) are not particularly limited.
  • the length of each complementary sequence between the three-dimensional region (D) and the binding region (A) is not particularly limited.
  • the length of each complementary sequence is, for example, 1 to 30 bases long, 1 to 10 bases long, or 1 to 7 bases long.
  • the three-dimensional formation region (D) and the binding region (A) may be directly or indirectly connected.
  • the direct connection means that, for example, the 3 ′ end of one region and the 5 ′ end of the other region are directly bonded, and the indirect connection is, for example, 3 of one region. It means that the “end” and the 5 ′ end of the other region are bonded via a linker region.
  • the intervening linker region may be, for example, a nucleic acid sequence or a non-nucleic acid sequence, preferably the former.
  • the length of the intervening linker region is not particularly limited and is, for example, 0 to 20 bases long, 1 to 10 bases long, or 1 to 6 bases long.
  • one end of the sensor (v) may be connected to the transistor.
  • the additional linker region may be further added to one end or both ends.
  • the length of the additional linker region is not particularly limited, and for example, the above description can be used.
  • one end of the sensor (v) may be connected to the transistor via the additional linker region.
  • the senor is a molecule including a nucleotide residue, and may be, for example, a molecule consisting of only a nucleotide residue or a molecule including a nucleotide residue.
  • the nucleotide is, for example, ribonucleotide, deoxyribonucleotide and derivatives thereof.
  • the sensor may be, for example, DNA containing deoxyribonucleotide and / or a derivative thereof, RNA containing ribonucleotide and / or a derivative thereof, or a chimera (DNA / RNA) containing the former and the latter But you can.
  • the sensor is preferably DNA.
  • the nucleotide may contain, for example, either a natural base (non-artificial base) or a non-natural base (artificial base) as a base.
  • a natural base include A, C, G, T, U, and modified bases thereof.
  • the modification include methylation, fluorination, amination, and thiolation.
  • the unnatural base include 2′-fluoropyrimidine, 2′-O-methylpyrimidine and the like. Specific examples include 2′-fluorouracil, 2′-aminouracil, 2′-O-methyluracil, And 2'-thiouracil.
  • the sensor is disposed in the transistor.
  • the sensor may be fixed directly or indirectly to the transistor.
  • the sensor is preferably fixed to the transistor at the end of the sensor.
  • the sensor may be fixed to the transistor via a fixing linker.
  • the linker may be, for example, a nucleic acid sequence or a non-nucleic acid sequence, and examples thereof include the above-described additional linker region.
  • the arrangement portion of the sensor can also be referred to as a detection portion in the transistor.
  • the non-nucleic acid sequence include sequences containing an alkyl group.
  • the sensor is immobilized on the transistor via the non-nucleic acid sequence, for example, as shown below.
  • n in (CH 2 ) n is, for example, 1 to 10, 2 to 10, or 6.
  • one end [—NH] of the non-nucleic acid sequence forms a single bond with an aldehyde group in the transistor, for example.
  • the other end [CH 2- ] of the non-nucleic acid sequence forms a single bond by an ester bond with phosphoric acid in the sensor, for example.
  • the immobilization method is not particularly limited, and examples thereof include chemical bonding.
  • the transistor has a structure in which Au (gold) is formed on an insulating film, a method of adding a thiol group to the sensor and immobilizing the sensor via the thiol group (T.TGoda and) Y.
  • the immobilization method for example, other known nucleic acid immobilization methods can be adopted. Examples of the method include a method using photolithography, and specific examples thereof can be referred to US Pat. No. 5,424,186.
  • the immobilization method includes, for example, a method of synthesizing the sensor on the transistor. As this method, for example, a so-called spot method can be mentioned.
  • US Pat. No. 5,807,522, Japanese Patent Publication No. 10-503841 and the like can be referred to.
  • the transistor is not particularly limited, and examples thereof include a transistor capable of detecting a change in charge in the Debye length range, and a specific example thereof is a field effect transistor.
  • a field effect transistor for example, a known field effect transistor can be used, and specific examples thereof include JP 2011-247795 A and International Publication No. 2014/024598.
  • the transistor includes, for example, a substrate, a source electrode, a drain electrode, a reference electrode, and a detection unit, and the source electrode, the drain electrode, and the detection unit are disposed on the substrate, and the detection The unit is disposed between the source electrode and the drain electrode, and the nucleic acid sensor is disposed in the detection unit.
  • the reference electrode is disposed in a solution containing an electrolyte (for example, a sample containing the electrolyte) that comes into contact with the detection unit.
  • the reference electrode is disposed so as not to contact the source electrode and the drain electrode.
  • the substrate, the source electrode, the drain electrode, and the like can refer to the configuration of the above-described known field effect transistor.
  • the transistor may include other configurations such as a gate electrode and an insulating film layer, for example, depending on the type of the field effect transistor.
  • the configuration of the aforementioned known field effect transistor can be referred to.
  • the device of the present invention may include, for example, a plurality of transistors.
  • each transistor includes a detection unit as described above, for example.
  • the number of sensors arranged in one detection unit is not particularly limited.
  • the Debye length means a Debye length of a solution (for example, an electrolyte solution) containing an electrolyte in contact with the transistor, and more specifically, a sample (for example, an electrolyte in contact with the detection unit of the transistor).
  • Debye length of a sample including The Debye length is not particularly limited, and can be calculated by a general Debye length calculation formula.
  • the Debye length can be calculated by the following formula (5).
  • the method for using the detection device of the present invention is not particularly limited, and can be used for the method for detecting a steroid-containing compound of the present invention as follows.
  • the sample is not particularly limited.
  • the sample may be, for example, either a sample containing the steroid compound or a sample in which it is unknown whether the steroid compound is contained.
  • the sample is preferably a liquid sample, for example.
  • the analyte when the analyte is a liquid, the analyte may be used as it is as a sample, or a diluted solution mixed in a solvent may be used as a sample.
  • the analyte is, for example, a solid or a powder, a mixed solution mixed with a solvent, a suspension suspended in a solvent, or the like may be used as a sample.
  • the solvent is not particularly limited, and examples thereof include water and a buffer solution.
  • the specimen examples include specimens collected from living organisms, soil, seawater, river water, sewage, food and drink, purified water, air, and the like.
  • the sample is preferably, for example, a liquid containing an electrolyte, that is, an electrolyte solution.
  • the sample can also be referred to as a sample containing an electrolyte.
  • the electrolyte is added to the sample.
  • the electrolyte is not particularly limited as long as it is a substance that separates into cations and anions in the liquid.
  • the contact step is a step of bringing a sample into contact with the detection device of the present invention.
  • the contact can be performed, for example, by bringing the sample into contact with the transistor in the detection device, and specifically, by bringing the sample into contact with a detection unit of the transistor.
  • the contact conditions (temperature, time) and the like in the contact step are not particularly limited.
  • the contact step may be performed, for example, by bringing the sample and the reagent into contact with the detection device, or by mixing the sample and the reagent in advance.
  • the mixture may be contacted.
  • the detection method of the present invention includes, for example, a mixing step of mixing the sample and the reagent, and a contacting step of bringing the mixture obtained into contact with the detection device.
  • the mixing is not particularly limited and can be performed by a known mixing method, for example, by bringing the reagent into contact with the sample.
  • the mixing conditions (temperature, time) and the like in the mixing step are not particularly limited.
  • the reagent include a reagent containing the first strand (ss1) or the second strand (ss2).
  • the detection step detects a steroid compound in the sample by detecting an increase or decrease in the number of nucleotide residues constituting the nucleic acid sensor in the range of the Debye length of the detection device.
  • the sensor increases or decreases the number of nucleotides of the Debye length as described above.
  • the nucleotide residue constituting the sensor has, for example, a negative charge. For this reason, in the presence of the steroid compound, the charge in the Debye length range is increased or decreased as compared to the absence of the steroid compound.
  • the detection step includes, for example, using the detection device to detect an increase or decrease in charge in the Debye length range, thereby increasing or decreasing the number of nucleotides in the Debye length, that is, a steroid in the sample.
  • Compounds can be detected. Therefore, the detection step includes, for example, a charge measurement step of measuring a charge in a Debye length range of the detection device using the detection device, and the Debye length based on the charge (measurement charge) and a reference charge.
  • a steroid compound detection step of detecting an increase or decrease in the number of nucleotide residues in a range and detecting the steroid compound.
  • the charge is measured by, for example, measuring an electric signal.
  • the electrical signal can be measured by, for example, a transistor of the detection device.
  • Examples of the electrical signal include a voltage and a current. Specific examples thereof include a voltage at the gate electrode (gate voltage), a current value at the drain electrode (drain current), and the like.
  • the current value at the drain electrode is preferably fixed.
  • the voltage at the gate electrode is, for example, a voltage when the current value at the drain electrode is 10 to 100 ⁇ A.
  • examples of the reference charge include charges in the Debye length range in the absence of the steroid compound. Then, by detecting whether the measured charge is increased or decreased compared to the reference charge, for example, the presence or absence of a steroid compound in the sample can be analyzed (qualitative), and the reference charge and the By detecting the difference in charge from the measured charge, for example, the amount of the steroid compound in the sample can be analyzed (quantified).
  • the presence of the steroid compound can be analyzed, and the reference charge and If it is the same or significantly higher than the reference charge, it can be analyzed without the steroid compound.
  • the number of nucleotide residues in the Debye length decreases due to the presence of the steroid compound, if the charge is significantly higher than the reference charge, the presence of the steroid compound can be analyzed and is the same as the reference charge, or the If it is significantly lower than the reference charge, it can be analyzed that there is no steroid compound.
  • the reference charge may be a calibration curve indicating a correlation between the amount of the steroid compound and the measured charge.
  • the amount of the steroid compound in the sample can be calculated based on the measured charge.
  • nucleic acid sensor (I) As the double-stranded nucleic acid sensor (nucleic acid sensor (I)), a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 (first strand) and a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 ( Second strand). In addition, a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 was synthesized as the single nucleic acid sensor (sensor (iii)). Next, on the gate insulating film such as SiO 2 or Si 3 N 4 in an ion sensitive transistor ISFET (see P.
  • the gate insulating film such as SiO 2 or Si 3 N 4 in an ion sensitive transistor ISFET (see P.
  • Organic monolayer was formed by self-organization method.
  • phosphoric acid in the first-strand and single-stranded nucleic acid sensors and the C-terminus via glutaraldehyde OHC (CH 2 ) 3 CHO which is an amine-reactive cross-linking agent A single bond was made with the amino terminus of the non-nucleic acid sequence represented by the above formula (4), which was ester-linked with.
  • n is 6.
  • the composition of the buffer was 5.558328 mmol / L KCl, 0.0588 mmol / L KH 2 PO 4 and 0.32408 mmol / L K 2 HPO 4 .
  • the conditions for measuring the Vg-ID characteristics were as follows.
  • Vg s Vg s
  • Vg 0 Vg
  • the double-stranded nucleic acid sensor is prepared by mixing 10 ⁇ L of a solution containing 10 ⁇ mol / L of the first strand and 10 ⁇ L of a solution containing 10 ⁇ mol / L of the second strand, and using the obtained mixed solution as the buffer. The mixture was added, incubated at 95 ° C. for 5 minutes, allowed to stand at room temperature for 55 minutes, and slowly returned to room temperature to form a double strand. Then, ⁇ Vg was calculated in the same manner as the single-stranded sensor except that the nucleic acid sensor after the double-stranded formation was immobilized on the ion-sensitive transistor ISFET as described above. These results are shown in FIGS.
  • FIG. 3 is a graph showing ⁇ Vg when the single-stranded sensor is used.
  • the horizontal axis indicates the concentration of cortisol
  • the vertical axis indicates ⁇ Vg.
  • ⁇ Vg increased with increasing cortisol concentration. That is, it was found that the concentration of cortisol can be measured based on ⁇ Vg.
  • FIG. 4 is a graph showing ⁇ Vg when the double-stranded sensor is used.
  • the horizontal axis indicates the concentration of cortisol
  • the vertical axis indicates ⁇ Vg.
  • ⁇ Vg decreased with increasing cortisol concentration. That is, it was found that the concentration of cortisol can be measured based on ⁇ Vg.
  • a part or all of the above embodiments and examples can be described as, but not limited to, the following supplementary notes (Appendix 1) Including a transistor in which a nucleic acid sensor for detecting a steroid skeleton-containing compound is disposed;
  • the nucleic acid sensor is A stereogenic region (D) that forms a predetermined stereostructure and a binding region (A) that binds to the steroid skeleton-containing compound; In the absence of the steroid skeleton-containing compound, the stereogenic region (D) is inhibited from forming the stereostructure, In the presence of the steroid skeleton-containing compound, by the contact of the steroid skeleton-containing compound with the binding region (A), the three-dimensional formation region (D) forms the three-dimensional structure, During the formation of the three-dimensional structure, The steroid skeleton-containing compound is present in a solution containing an electrolyte, A device for detecting a steroid skeleton-containing compound, wherein the
  • the first strand (ss1) has the three-dimensional region (D) and the binding region (A) in this order
  • the second strand (ss2) has a stem forming region (S D ) and a stem forming region (S A ) in this order
  • the stem forming region (S D ) has a sequence complementary to the three-dimensional forming region (D)
  • the stem forming region (S A ) has a sequence complementary to the binding region (A)
  • the stereogenic region (D) is inhibited from forming the stereostructure and hybridizes with the second chain (ss2)
  • the three-dimensional formation region (D) forms the three-dimensional structure by contacting the steroid skeleton-containing compound with the binding region (A) of the first chain (ss1), and Dis
  • (II) a single-stranded nucleic acid sensor having the three-dimensional formation region (D) and the binding region (A), In the absence of the steroid skeleton-containing compound, the stereogenic region (D) is inhibited from forming the stereostructure, In the presence of the steroid skeleton-containing compound, by the contact of the steroid skeleton-containing compound with the binding region (A), the three-dimensional formation region (D) forms the three-dimensional structure, During the formation of the three-dimensional structure, A single-stranded nucleic acid sensor, wherein the number of nucleotide residues constituting the nucleic acid sensor in the Debye length range is greater than that at the time of inhibiting the formation of the three-dimensional structure.
  • nucleic acid sensor (II) is at least one nucleic acid sensor selected from the group consisting of the following (i) to (iv) and (v).
  • the blocking region (B) is complementary to a partial region (Dp) in the three-dimensional region (D);
  • a terminal region (Ab) on the blocking region (B) side in the binding region (A) is complementary to a region (Df) adjacent to the partial region (Dp) in the three-dimensional formation region (D), and
  • a single-stranded nucleic acid sensor which is complementary to a terminal region (Af) opposite to the blocking region (B) in the binding region (A).
  • a single-stranded nucleic acid sensor having the first region (D1) on one end side of the binding region (A) and the second region (D2) on the other end side of the binding region (A) .
  • (V) having the three-dimensional formation region (D) and the binding region (A) in this order;
  • the single-stranded nucleic acid sensor in which the three-dimensional region (D) and the binding region (A) have complementary sequences.
  • (Appendix 9) In the above-mentioned (i) or (ii) single-stranded nucleic acid sensor, The detection device according to supplementary note 8, including the three-dimensional formation region (D), the blocking region (B), and the binding region (A) in this order from the 5 ′ side.
  • the first region (D1) and the second region (D2) are: The detection device according to appendix 8, wherein the detection devices each include a sequence complementary to each other at the end opposite to the position of the binding region (A).
  • the detection device according to appendix 8 In the (v) single-stranded nucleic acid sensor, The detection device according to appendix 8, wherein the sequence from the 5 'side of the three-dimensional formation region (D) and the sequence from the 3' side of the binding region (A) have complementary sequences to each other.
  • the three-dimensional formation region (D) is a G formation region (G) forming a G-quartet structure, 15.
  • Appendix 18 A contact step of bringing a sample into contact with the detection device according to any one of appendices 1 to 17, and detecting an increase or decrease in the number of nucleotide residues constituting the nucleic acid sensor in the range of the Debye length of the detection device.
  • skeleton containing compound in the said sample is included, The detection method of the steroid frame
  • Appendix 19 Using the detection device described in Appendix 5, A mixing step of mixing the sample and the reagent; Contacting the resulting mixture with the detection device, and detecting the increase or decrease in the number of nucleotide residues constituting the nucleic acid sensor in the Debye length range of the detection device, thereby the steroid in the sample
  • the detection step comprises A charge measuring step of measuring a charge in a debye length range of the detection device by the detection device; Supplementary note 18 or 19, comprising detecting an increase or decrease in the number of the nucleotide residues in the Debye length range based on the charge and the reference charge, and detecting the steroid skeleton-containing compound.
  • the detection method described. The detection method according to appendix 20, wherein the charge measurement is an electric signal measurement.
  • a steroid skeleton-containing compound containing cortisol can be detected.
  • the present invention can be said to be an extremely useful technique for research and examination in various fields such as clinical medicine, food, and environment.

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Abstract

L'invention concerne un nouveau dispositif de détection de composé contenant un squelette stéroïde et un procédé de détection de composé contenant un squelette stéroïde l'utilisant. Un dispositif de détection de composé contenant un squelette stéroïde selon la présente invention est caractérisé en ce qu'il comprend un transistor sur lequel est disposé un capteur d'acide nucléique pour détecter un composé contenant un squelette stéroïde, et est caractérisé en ce que le capteur d'acide nucléique comprend : une région de formation de corps tridimensionnel (D) qui forme une structure tridimensionnelle prédéterminée ; et une région de liaison (A) qui se lie au composé contenant un squelette stéroïde, la région de formation de corps tridimensionnel (D) inhibe la formation de la structure tridimensionnelle en l'absence du composé contenant un squelette stéroïde, la région de formation de corps tridimensionnel (D) forme la structure tridimensionnelle suite au contact du composé contenant un squelette stéroïde avec la région de liaison (A) en présence du composé contenant un squelette stéroïde, et au moment de la formation de la structure tridimensionnelle, le composé contenant un squelette stéroïde est présent dans une solution contenant un électrolyte, et le nombre de résidus nucléotidiques constituant le capteur d'acide nucléique dans la solution dans la plage de sa longueur de Debye augmente ou diminue par rapport à ce qui est observé lorsque la formation de la structure tridimensionnelle est inhibée.
PCT/JP2017/034996 2017-03-27 2017-09-27 Dispositif de détection de composé contenant un squelette stéroïde et procédé de détection de composé contenant un squelette stéroïde l'utilisant WO2018179514A1 (fr)

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Publication number Priority date Publication date Assignee Title
JP7482795B2 (ja) 2018-12-21 2024-05-14 イルミナ インコーポレイテッド 感知システム
JP2021032607A (ja) * 2019-08-20 2021-03-01 学校法人早稲田大学 アプタマー固定化半導体センシングデバイス及び非荷電分子の検出方法
JP2021076529A (ja) * 2019-11-12 2021-05-20 学校法人早稲田大学 アプタマー固定化半導体センシングデバイス及び非荷電分子の検出方法
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