WO2016159197A1 - Rna aptamer and sensor using same - Google Patents

Abstract

[Problem] To provide an RNA aptamer that binds specifically to HbA1c and a sensor using the same. [Solution] An RNA aptamer 130 having a common sequence AGNNNWGGWSC (SEQ ID NO:1) (excluding a base sequence wherein N's at the 3-5th positions from the 5' end are respectively adenine, guanine and guanine), AGNNNNWGGWSC (SEQ ID NO:2), AGNNNNNWGGWSC (SEQ ID NO:3) or AUGNNNAGGACC (SEQ ID NO:4) [wherein: N is adenine, guanine, cytosine or uracil; W is adenine or uracil; and S is guanine or cytosine] and being capable of binding to HbA1c.

Description

RNA aptamer and sensor using the same

The present invention relates to an RNA aptamer and a sensor using the RNA aptamer.

An antibody for measuring hemoglobin contained in a sample liquid using a detection element and a sensor using the same are known (for example, see Patent Document 1 or 2).

JP 2002-209579 A Japanese Patent Laid-Open No. 06-1113830

Since the antibody used in Patent Document 1 or 2 is produced using animal cells, variation between batches due to individual differences occurs, and it is not suitable for mass production by chemical synthesis, and it is difficult to reduce production costs. Furthermore, since an antibody is a protein, it has a property of being easily denatured under high temperature or a dry environment and having poor storage stability.

Therefore, an aptamer that specifically binds to hemoglobin, has low quality variation, and is low in cost and excellent in storage stability has been demanded.

The RNA aptamer according to the embodiment of the present invention includes AGNNNNGWGWSC (SEQ ID NO: 1) (excluding nucleotide sequences in which the third to fifth Ns counted from the 5 ′ end are adenine, guanine, and guanine, respectively), AGNNNNWGGWSC (sequence No. 2), AGNNNNNNGWGWSC (SEQ ID NO: 3), or AUGNNNNAGACC (SEQ ID NO: 4), the consensus sequence (N is adenine, guanine, cytosine or uracil, W is adenine or uracil, S is guanine or cytosine Included).

A sensor according to an embodiment of the present invention includes a base and the above-described RNA aptamer immobilized on the base.

According to the RNA aptamer according to the embodiment of the present invention, by having the configuration as described above, it effectively binds specifically to at least HbA1c among the fraction components of hemoglobin (hereinafter also referred to as Hb). It becomes possible.

According to the sensor according to the embodiment of the present invention, it is possible to effectively specifically detect at least HbA1c among the fraction components of hemoglobin by having the above-described configuration.

It is a figure which shows the RNA aptamer which has a base sequence shown by sequence number 88, (a) shows a primary structure, (b) shows a secondary structure. It is a figure which shows the secondary structure of RNA aptamer, (c) shows the secondary structure of RNA aptamer which has a base sequence shown by sequence number 95, (d) is RNA which has the base sequence shown by sequence number 96 The secondary structure of an aptamer is shown, (e) shows the secondary structure of the RNA aptamer which has a base sequence shown by sequence number 97. It is a figure which shows the sensor which concerns on embodiment of this invention, (a) is a top view, (b) is sectional drawing of a length direction, (c) is sectional drawing of the width direction. It is sectional drawing which expands and shows a part of sensor of FIG. It is a top view which shows the detection element of the sensor of FIG. FIG. 3 is an exploded plan view of the sensor of FIG. 2. It is a figure which shows the manufacturing process of the sensor of FIG. FIG. 6 is a plan view showing a modified example of the sensor in FIG. 2, (a) and (b) corresponding to FIG. 6 (d), and (c) corresponding to FIG. 2 (a). It is a figure which shows the modification of the sensor of FIG. 2, (a) is a top view, (b) is sectional drawing of a length direction, (c) is sectional drawing of the width direction. It is a figure which shows the manufacturing process of the sensor of FIG. It is a figure which shows the modification of the sensor of FIG. 2, and is a figure which shows a manufacturing process especially. It is a top view which shows the modification of the sensor of FIG. 2, and is a figure corresponding to FIG.6 (d). It is a side view which shows the Example of the detection part of the sensor of FIG. It is a side view which shows the Example of the detection part of the sensor of FIG. It is a figure which shows the experimental data regarding the sensor (RNA aptamer) which concerns on embodiment of this invention.

Hereinafter, embodiments of an RNA aptamer and a sensor using the RNA aptamer according to an embodiment of the present invention will be described in detail with reference to the drawings. In addition, in each drawing demonstrated below, the same code shall be attached | subjected to the same structural member. Further, the size of each member, the distance between members, and the like are schematically illustrated, and may differ from actual ones.

<RNA aptamer>
RNA aptamer 130 according to an embodiment of the present invention includes AGNNNNWGGWSC (SEQ ID NO: 1) (excluding nucleotide sequences in which the third to fifth Ns counted from the 5 ′ end are adenine, guanine, and guanine, respectively), AGNNNNWGGWSC ( SEQ ID NO: 2), AGNNNNNNGWGWSC (SEQ ID NO: 3), or AUGNNNNAGACC (SEQ ID NO: 4), a consensus sequence (N is adenine, guanine, cytosine or uracil, W is adenine or uracil, S is guanine or cytosine And has a binding ability to HbA1c. By having such a configuration, the RNA aptamer 130 of the present invention can specifically bind to HbA1c among the Hb fraction components. The RNA aptamer 130 of the present invention does not bind to human serum albumin (HSA) and human IgG (h-IgG).

In one embodiment of the present invention, the RNA aptamer 130 has a common sequence represented by AGNHNWGGWSC (SEQ ID NO: 5), AGNHNNWGGWSC (SEQ ID NO: 6), AGNHNNNNGWGWSC (SEQ ID NO: 7), or AUGNHNAGGACC (SEQ ID NO: 8). (N is adenine, guanine, cytosine or uracil, H is adenine, cytosine or uracil, W is adenine or uracil, and S is guanine or cytosine).

In one embodiment of the present invention, the RNA aptamer 130 has a consensus sequence of AGNNNNGWGWSC (SEQ ID NO: 1) (base sequence in which the 3rd to 5th N from the 5 ′ end are adenine, guanine, and guanine, respectively. Except), AGNNNNNGWGWSC (SEQ ID NO: 2), or a base sequence represented by AGNNNNNNGWGWSC (SEQ ID NO: 3) (N is adenine, guanine, cytosine or uracil, W is adenine or uracil, and S is guanine or cytosine) ).

In one embodiment of the present invention, the common sequence of the RNA aptamer 130 is a base sequence represented by AGNNNNAGACC (SEQ ID NO: 9) (N is adenine, guanine, cytosine or uracil, and 3 to 5 counted from the 5 ′ end. N may be a base sequence excluding adenine, guanine, and guanine, respectively.

In one embodiment of the present invention, the RNA aptamer 130 has a common sequence represented by AGWYDAGGACC (SEQ ID NO: 10) (W is adenine or uracil, Y is uracil or cytosine, D is adenine, guanine) Or uracil).

In one embodiment of the present invention, the common sequence of the RNA aptamer 130 is AGACGAGGACC (SEQ ID NO: 11), AGAUUAGGACC (SEQ ID NO: 12), AGACAAGGACC (SEQ ID NO: 13), AGAUGAGGAACC (SEQ ID NO: 14), AGUCGAGGACC (SEQ ID NO: 15). ), AGACUAGGACC (SEQ ID NO: 16), AGAUUGGAGGAGC (SEQ ID NO: 17), AGAGAGGGUGC (SEQ ID NO: 18), AGGAAUGGACC (SEQ ID NO: 19), AGAAUUGAGGACC (SEQ ID NO: 20), AGAACGAGGACC (SEQ ID NO: 21), or AUGAUUAGGACC (SEQ ID NO: 21) ). The common sequence of the RNA aptamer 130 is AGACGAGGACC (SEQ ID NO: 11), AGAUUAGGACC (SEQ ID NO: 12), AGACAAGGACC (SEQ ID NO: 13), AGAUGAGGACC (SEQ ID NO: 14), AGUCGAGGACC (SEQ ID NO: 15), or AGACUAGGACC (SEQ ID NO: 15). The base sequence shown by number 16) may be sufficient.

In one embodiment of the invention, the RNA aptamer 130 may have a fluorine group at the 2 ′ position of ribose in at least one nucleotide in the consensus sequence, and a fluorine at the 2 ′ position of adenine and uracil ribose in the consensus sequence. It may have a group. According to this, the stability of the structure of the RNA aptamer 130 can be improved. The RNA aptamer 130 may have a fluorine group at the 2'-position of the ribose of nucleotides other than the nucleotides contained in the common sequence.

In one embodiment of the present invention, the total number of nucleotides of the RNA aptamer 130 is not particularly limited as long as the RNA aptamer 130 has a binding ability to HbA1c, but may be 23 to 96.

The RNA aptamer 130 according to one embodiment of the present invention can be prepared by a known method as long as it has the sequence as described above. For example, it can be prepared by a known chemical synthesis method. it can.

As an example of the primary structure of the RNA aptamer 130 according to this embodiment, the primary structure of an RNA aptamer having the base sequence represented by SEQ ID NO: 88 is shown in FIG. The RNA aptamer 130 includes a common sequence region 130a including a common sequence, a first nucleotide sequence region 130b1 including a first nucleotide sequence that binds to the 5 ′ end of the common sequence, and a second nucleotide that binds to the 3 ′ end of the common sequence. A second nucleotide sequence region 130b2 containing the sequence and a terminal region 130c (5 ′ end 130c1 and 3 ′ end 130c2) are provided.

When the secondary structure of the RNA aptamer 130 having the base sequence represented by SEQ ID NO: 88 is displayed using the software for predicting the secondary structure (Centroid Fold, M. Hamada et al .: Bioinformatics, 2008 Dec. 18) The shape is as shown in FIG. In the RNA aptamer, cytosine and guanine in the common sequence region 130a form a base pair, and the common sequence region forms a loop structure. The nucleotides contained in the first nucleotide sequence region 130b1 and the nucleotides contained in the second nucleotide sequence region 130b2 form a base pair to form a stem region 130b. That is, the RNA aptamer 130 according to the present embodiment has a so-called stem loop shape, and the common sequence region 130a is included in the loop structure.

In the present specification, “stem loop shape” or “stem loop structure” is a concept that occurs when a secondary structure of a base sequence is shown, and single-stranded RNA forms a partially complementary base pair. The other part has a loop structure including one or more base pairs and a single-stranded base sequence, and the 5 ′ end and 3 ′ end of the loop structure are directly linked to the stem structure. Represents the structure. The “stem structure” may include a base pair mismatch site, a bulge structure, and the like.

When the secondary structure of the RNA aptamer 130 having the base sequences shown by SEQ ID NOs: 95 to 97 is displayed using the software (Centroid Fold) for predicting the secondary structure, it is shown in FIGS. 1B (c) to (e), respectively. The shape is as shown. In the RNA aptamer 130 having the base sequence represented by SEQ ID NO: 95, the common sequence region 130a is located in a loop structure consisting of 15 nucleotides without forming a base pair, and the six nucleotides contained in the first nucleotide sequence region 130b1 And the six nucleotides included in the second nucleotide sequence region 130b2 form base pairs to form the stem region 130b. In the RNA aptamer having the base sequence represented by SEQ ID NO: 96, the common sequence region 130a has a loop structure consisting of three base pairs of six nucleotides contained in the region and five nucleotides including a single-stranded structure. The nine nucleotides included in the first nucleotide sequence region 130b1 and the nine nucleotides included in the second nucleotide sequence region 130b2 form base pairs to form the stem region 130b. In the RNA aptamer having the base sequence represented by SEQ ID NO: 97, the common sequence region 130a is located in a loop structure consisting of 19 nucleotides without forming a base pair, and the six nucleotides contained in the first nucleotide sequence region 130b1 The six nucleotides contained in the second nucleotide sequence region 130b2 form base pairs to form the stem region 130b.

(Common array area)
The common sequence region 130a has one base sequence selected from the base sequences shown in SEQ ID NOs: 1 to 22. In an embodiment of the present invention, the common array region 130a may form a loop structure. The loop structure consists of one of guanine present from the first to the third position from the 5 ′ end of the common sequence and one of cytosine present from the first position to the third position from the 3 ′ end of the common sequence. One may be formed by forming a base pair.

(Stem area)
In one embodiment of the present invention, the RNA aptamer 130 includes a first nucleotide sequence region 130b1 that binds to the 5 ′ end of the common sequence, and a second nucleotide sequence region 130b2 that binds to the 3 ′ end of the common sequence. The stem region 130b may be formed by forming a base pair between the nucleotide contained in the one nucleotide sequence region 130b1 and the nucleotide contained in the second nucleotide sequence region 130b2. In the stem region 130b, the first nucleotide sequence region 130b1 includes at least one nucleotide that forms a base pair with the nucleotide in the second nucleotide sequence region 130b2. The number of nucleotides forming such a base pair is not particularly limited, but may be 1 to 13, or 6 to 13. If the number of nucleotides forming the base pair is 6 or more, the binding ability between the RNA aptamer and HbA1c can be improved.

The base pair formed may be a base pair of cytosine and guanine, or a base pair of adenine and uracil. At least one of the base pairs may be a base pair of cytosine and guanine. In addition, by increasing the number of base pairs of cytosine and guanine in the formed base pair, the stability of the stem region 130b can be improved, and the binding ability of the RNA aptamer to HbA1c can be improved.

In one embodiment of the present invention, the first nucleotide sequence region 130b1 or the second nucleotide sequence region 130b2 can include at least one nucleotide that forms a base pair with a nucleotide in the common sequence. The number of nucleotides forming such a base pair is not particularly limited, but may be 1 to 9, or 6 to 9. If the number of nucleotides forming the base pair is 6 or more, the binding ability between the RNA aptamer and HbA1c can be improved. The base pair may be a base pair of cytosine and guanine, or a base pair of adenine and uracil. At least one of the base pairs may be a base pair of cytosine and guanine. In addition, by increasing the number of base pairs of cytosine and guanine in the formed base pair, the stability of the stem region 130b can be improved, and the binding ability of the RNA aptamer to HbA1c can be improved.

(Terminal region)
In one embodiment of the present invention, the terminal region 130c may have a modifying group such as biotin, a thiol group, or an amino group. The terminal region 130c means both ends of the base sequence of the RNA aptamer 130 and has a 3 ′ end 130c1 and a 5 ′ end 130c2. When the terminal region 130c has a modifying group, it becomes easy to immobilize the RNA aptamer 130 to another member. Here, each of the above-described modifying groups can be bonded to at least one of the 3 ′ end 130c1 and the 5 ′ end 130c2 in the end region 130c. For example, by binding at least one of the above-described modifying groups to both ends, it is possible to increase the variation of immobilization or improve the immobilization property of immobilization.

Here, when the terminal region 130c has biotin, streptavidin (hereinafter sometimes referred to as SA) may be bound to the biotin. According to this, the immobilization of RNA aptamer 130 on other members is facilitated by an amide bond with streptavidin.

Further, when the terminal region 130c has a thiol group, it can be bonded to gold by a gold-thiol bond. Therefore, the RNA aptamer 130 can be relatively easily immobilized to other members via gold bonded to the thiol group. In the case where the terminal region 130 c has a thiol group, a spacer having a predetermined length can be interposed between the thiol group and the RNA aptamer 130. According to this, the detection sensitivity of the detection target by the RNA aptamer 130 can be improved by appropriately adjusting the height from the base on which the RNA aptamer 130 is immobilized to the RNA aptamer 130.

In addition, when the terminal region 130c has an amino group, the terminal region 130c may have a first substance that forms an amide bond with the amino group. Here, examples of the first substance include polyethylene glycol (PEG). At this time, the amino group forms an amide bond with the carboxyl group modified at the end of polyethylene glycol. Then, the RNA aptamer 130 can be immobilized with another member via a thiol group modified at the other end of polyethylene glycol.

<Sensor>
A sensor 100 according to an embodiment of the present invention will be described with reference to FIGS.

The sensor 100 according to the present embodiment mainly includes a first cover member 1, an intermediate cover member 1A, a second cover member 2, and a detection element 3, as shown in FIG. In FIG. 2A, the vicinity of the detection element 3 is partially shown through the second cover member 2 for easy understanding of the structure.

Specifically, as shown in FIG. 2B, the sensor 100 includes an inflow portion 14 into which the sample liquid flows, an inflow portion 14, and an intermediate cover member 1 </ b> A and a second cover member 2. And a flow path 15 that is surrounded and extends to at least the detection unit 13. FIG. 2C shows a cross-sectional view of FIG. 2A, showing an aa cross section, a bb cross section, and a cc cross section in order from the top. As shown in FIG. 2B, the inflow portion 14 is provided so as to penetrate the second cover member 2 in the thickness direction. The inflow portion 14 may be provided so as to be located on the side surfaces of the intermediate cover member 1 </ b> A and the second cover member 2.

In the sensor 100 according to the present embodiment, since the detection element and the intermediate cover member that constitutes at least a part of the flow path are provided on the upper surface of the first cover member, the inflow portion even when a thick detection element is used. It is possible to secure a flow path for the sample liquid from the detection part to the detection part, and to allow the sample liquid sucked from the inflow part to flow to the detection part by capillary action or the like. That is, it is possible to provide a sensor with a simple measuring operation that includes a specimen liquid suction mechanism while using a detecting element having a thickness. Further, in the flow path of the sample liquid, the contact angles θ1a and θ2a with respect to the sample liquid on the surface of the member positioned upstream of the detection element are made smaller than the contact angle θ3 with respect to the sample liquid on the surface of the detection element. Thus, the sample liquid flowing in from the inflow portion can flow smoothly toward the detection element (detection portion) through the surface of the member located on the upstream side.

(First cover member 1)
The first cover member 1 has a flat plate shape as shown in FIG. The thickness is, for example, 0.1 mm to 0.5 mm. The planar shape of the first cover member 1 is generally rectangular. The length of the first cover member 1 in the length direction is, for example, 1 cm to 5 cm, and the length in the width direction is, for example, 1 cm to 3 cm. As a material of the first cover member 1, for example, paper, plastic, celluloid, ceramics, nonwoven fabric, glass, or the like can be used. Plastics may be used from the viewpoint of combining required strength and cost.

Further, as shown in FIG. 2A, the upper surface of the first cover member 1 is formed with a terminal 6 and a wiring 7 routed from the terminal 6 to the vicinity of the detection element 3. The terminals 6 are formed on both sides of the detection element 3 in the width direction on the upper surface of the intermediate cover member 1A. When measuring the sensor 100 with an external measuring instrument (not shown), the terminal 6 and the external measuring instrument are electrically connected. Further, the terminal 6 and the detection element 3 are electrically connected through a wiring 7 or the like. Then, a signal from an external measuring instrument is input to the sensor 100 via the terminal 6, and a signal from the sensor 100 is output to the external measuring instrument via the terminal 6.

(Intermediate cover member 1A)
In the present embodiment, as shown in FIG. 2A, the intermediate cover member 1 </ b> A is positioned alongside the detection element 3 on the upper surface of the first cover member 1. Further, the intermediate cover member 1A and the detection element 3 are located via a gap.

The intermediate cover member 1A has a flat frame shape having a concave portion 4 on a flat plate, and has a thickness of, for example, 0.1 mm to 0.5 mm.

In the present embodiment, the recess forming portion 4 is a portion that divides the first upstream portion 1Aa and the first downstream portion 1Ab as shown in FIG. By joining the intermediate cover member 1A provided with the recessed portion forming portion 4 to the flat first cover member 1, the element housing recessed portion 5 is formed by the first cover member 1 and the intermediate cover member 1A. . That is, the upper surface of the first cover member 1 positioned inside the recess forming portion 4 is the bottom surface of the element housing recess 5, and the inner wall of the recess forming portion 4 is the inner wall of the element housing recess 5.

As the material of the intermediate cover member 1A, for example, resin (including plastic), paper, non-woven fabric, and glass can be used, and more specifically, resin materials such as polyester resin, polyethylene resin, acrylic resin, and silicone resin. May be used. The material of the first cover member 1 and the material of the intermediate cover member 1A may be the same or different.

Further, in the present embodiment, the intermediate cover member 1A has a first upstream portion 1Aa and a first downstream portion 1Ab. As shown in FIG. It is located between the first upstream portion 1Aa and the first downstream portion 1Ab. According to this, the amount of the sample liquid flowing on the detection element 3 through the first upstream portion 1Aa in the flow path 15 exceeds the amount necessary for measurement flows to the first downstream portion 1Ab side. An appropriate amount of sample liquid can be supplied to the detection element 3.

Note that the thickness of the intermediate cover member 1 </ b> A may be larger than the thickness of the detection element 3.

(Second cover member 2)
As shown in FIG. 2B, the second cover member 2 covers at least a part of the detection element 3 and is joined to the intermediate cover member 1A. As a material of the second cover member 2, for example, resin (including plastic), paper, non-woven fabric, and glass can be used, and more specifically, a resin such as a polyester resin, a polyethylene resin, an acrylic resin, and a silicone resin. Materials may be used. The material of the first cover member 1 and the material of the second cover member 2 may be the same. As a result, it is possible to suppress the deformation caused by the difference between the thermal expansion coefficients of each other. The second cover member 2 may be configured to be joined only to the intermediate cover member 1A, or may be joined to both the first cover member 1 and the intermediate cover member 1A.

Here, the second cover member 2 has a third substrate 2a and a fourth substrate 2b.

The third substrate 2a is bonded to the upper surface of the intermediate cover member 1A. The third substrate 2a is flat and has a thickness of, for example, 0.1 mm to 0.5 mm. The fourth substrate 2b is bonded to the upper surface of the third substrate 2a. The fourth substrate 2b is flat and has a thickness of 0.1 mm to 0.5 mm, for example. Then, by joining the fourth substrate 2b to the third substrate 2a, the flow path 15 is formed in the lower portion of the second cover member 2 as shown in FIG. The flow path 15 extends from the inflow part 14 to at least a region immediately above the detection part 13, and the cross-sectional shape is, for example, a rectangular shape.

In the present embodiment, as shown in FIG. 2B, the end portion of the flow path 15 is configured such that the gap between the fourth substrate 2b and the intermediate cover member 1A functions as the exhaust hole 18 without the third substrate 2a. To do. The exhaust hole 18 is for releasing air in the flow path 15 to the outside. The exhaust hole 18 may have any shape such as a columnar shape or a quadrangular prism shape as long as the air in the flow path 15 can be extracted. However, if the opening of the exhaust hole 18 is too large, the area where the sample liquid existing in the flow channel 15 comes into contact with the outside air becomes large, and the moisture of the sample liquid is likely to evaporate. If it does so, the density | concentration change of a sample liquid will occur easily and it will cause the fall of a measurement precision. Therefore, the opening of the exhaust hole 18 may be set so as not to be larger than necessary. Specifically, in the case of the exhaust hole 18 made of a columnar shape, the diameter thereof is set to 1 mm or less, and in the case of the exhaust hole 18 made of a square column, one side thereof is set to be 1 mm or less. Further, the inner wall of the exhaust hole 18 is hydrophobic. As a result, the sample liquid filled in the flow path 15 is prevented from leaking out from the exhaust hole 18.

The first cover member 1, the intermediate cover member 1A, and the second cover member 2 can all be formed of the same material. According to this, since the thermal expansion coefficients of the respective members can be substantially uniformed, deformation due to the difference in the thermal expansion coefficients of the respective members is suppressed. In addition, a biomaterial may be applied to the detection unit 13, but some of them may be easily altered by external light such as ultraviolet rays. In that case, as the material for the first cover member 1, the intermediate cover member 1 </ b> A, and the second cover member 2, an opaque material having a light shielding property may be used. On the other hand, when almost no alteration due to light outside the detection unit 13 occurs, the second cover member 2 constituting the flow path 15 may be formed of a material close to transparency. In this case, the state of the sample liquid flowing in the flow channel 15 can be visually confirmed.

(Detecting element 3)
As shown in FIG. 2B, the detection element 3 is a base 10 positioned on the top surface of the first cover member 1, and a detection target that is positioned on the top surface of the base 10 and contained in the sample liquid. It has at least one detection part 13 which performs. Details of the detection element 3 are shown in FIG. 3B and FIG.

In the present embodiment, as shown in FIG. 4, an electrode pattern is provided on the upper surface of the substrate 10, and an insulating member 28 shown in FIG. 8 is provided so as to cover the electrode pattern as necessary. May be. The electrode pattern corresponds to an IDT (Inter Digital Transducer) electrode when a SAW element is used as the detection element 3. In the present embodiment, a first IDT electrode 11, a second IDT electrode 12, a first extraction electrode 19, a second extraction electrode 20, and the like, which will be described later, are provided on the upper surface of the substrate 10. In the present embodiment, as shown in FIG. 3B, the second cover member 2 is fixed on the IDT electrodes 11 and 12, for example, on the upper surface of the base 10.

(Substrate 10)
The substrate 10 is made of, for example, a single crystal substrate having piezoelectricity such as a lithium tantalate (LiTaO ₃ ) single crystal, a lithium niobate (LiNbO ₃ ) single crystal, or quartz. The planar shape and various dimensions of the substrate 10 may be set as appropriate. As an example, the thickness of the substrate 10 is 0.3 mm to 1 mm.

(IDT electrodes 11, 12)
As shown in FIG. 4, the first IDT electrode 11 has a pair of comb electrodes. Each comb electrode has two bus bars facing each other and a plurality of electrode fingers extending from each bus bar to the other bus bar side. The pair of comb electrodes are arranged so that a plurality of electrode fingers mesh with each other. The second IDT electrode 12 is configured similarly to the first IDT electrode 11. The first IDT electrode 11 and the second IDT electrode 12 constitute a transversal IDT electrode.

The first IDT electrode 11 is for generating a predetermined surface acoustic wave (SAW: Surface Acoustic Wave), and the second IDT electrode 12 is for receiving the SAW generated by the first IDT electrode 11. The first IDT electrode 11 and the second IDT electrode are arranged in the same straight line so that the second IDT electrode 12 can receive the SAW generated in the first IDT electrode 11. The frequency characteristics can be designed using parameters such as the number of electrode fingers of the first IDT electrode 11 and the second IDT electrode 12, the distance between adjacent electrode fingers, and the cross width of the electrode fingers. As the SAW excited by the IDT electrode, there are various vibration modes. In the detection element 3 according to the present embodiment, for example, a transverse wave vibration mode called an SH wave is used.

An elastic member for suppressing SAW reflection may be provided outside the first IDT electrode 11 and the second IDT electrode 12 in the SAW propagation direction (width direction). The SAW frequency can be set, for example, within a range of several megahertz (MHz) to several gigahertz (GHz). In particular, if it is several hundred MHz to 2 GHz, it is practical, and downsizing of the detection element 3 and thus downsizing of the sensor 100 can be realized.

(Extraction electrodes 19, 20)
As shown in FIG. 4, the first extraction electrode 19 is connected to the first IDT electrode 11, and the second extraction electrode 20 is connected to the second IDT electrode 12. The first extraction electrode 19 is extracted from the first IDT electrode 11 to the opposite side of the detection unit 13, and the end 19 e of the first extraction electrode 19 is electrically connected to the wiring 7 provided on the first cover member 1. Yes. The second extraction electrode 20 is extracted from the second IDT electrode 12 to the side opposite to the detection unit 13, and the end 20 e of the second extraction electrode 20 is electrically connected to the wiring 7.

Here, the first IDT electrode 11, the second IDT electrode 12, the first extraction electrode 19 and the second extraction electrode 20 are made of, for example, aluminum or an alloy of aluminum and copper. These electrodes may have a multilayer structure. In the case of a multilayer structure, for example, titanium or chromium can be included in the first layer, and aluminum or aluminum alloy can be included in the second layer.

(Detector 13)
As shown in FIG. 4, the detection unit 13 is located on the surface of the substrate 10 and on the propagation path of the elastic wave propagating from the first IDT electrode 11 to the second IDT electrode 12. Hereinafter, an example using the RNA aptamer 130 according to this embodiment described above will be described.

For example, as shown in FIG. 12A, the detection unit 13 includes a plurality of SAs 134 immobilized on the substrate 10, a plurality of biotins 131a respectively bonded to the plurality of SAs 134, and a plurality of biotins 131a. And a plurality of RNA aptamers (binding portions) 130 that are respectively bonded through the main chain 133. That is, the RNA aptamer (binding portion) 130 has biotin 131a via a double strand 133 in the terminal region 130c. The double strand 133 has a configuration in which AAAAAAAAAAAAAAAAAAA (SEQ ID NO: 23) added to the 3 ′ end of the RNA aptamer 130 and TTTTTTTTTTTTTTTTTTT (SEQ ID NO: 24) bound to biotin 131a form a base pair. .

In addition, as a modification of the detection unit 13, a configuration as shown in FIG.

In this modification, the detection unit 13 includes an immobilization film 13a positioned on the surface of the substrate 10, a chain substance (SAM) 13c that is bonded to the immobilization film 13a, and a plurality of bonds that are bonded to the SAM. , A plurality of biotins 131a respectively binding to a plurality of SA134s, and a plurality of RNA aptamers (binding portions) 130 respectively binding to a plurality of biotins 131a. Here, for example, Au (gold), Ti, Cu, or the like can be used as the material forming the fixing film 13a, and Au may be used. The immobilization film 13a may be, for example, a gold film or a gold two-layer structure formed on chromium. Further, a protective film may be interposed between the base 10 and the immobilizing film 13a. Examples of the chain substance 13c include alkane, polyethylene glycol, or a complex molecule of alkane and polyethylene glycol.

Further, as a modification of the detection unit 13, a configuration as shown in FIG.

In this modification, the RNA aptamer 130 of the detection unit 13 has an amino group in the terminal region 130c. In this case, the first substance 132 (chain substance 13c) that forms an amide bond with the amino group 131c may be included. Here, examples of the first substance 132 include polyethylene glycol (PEG). At this time, the amino group 131c forms an amide bond with the modified carboxyl group at the end of polyethylene glycol. And it can fix | immobilize with another member via the thiol group 131b modified by the other terminal of polyethyleneglycol. Specifically, as shown in FIG. 13A, the detection unit 13 includes an immobilization film (Au) 13a located on the surface of the substrate 10 and a plurality of chains bonded to the immobilization film 13a. And a plurality of RNA aptamers (binding portions) 130 that are respectively bonded to the plurality of chain substances 13c via amino groups 131c. Then, the carboxyl group located at the end of polyethylene glycol may be active esterified and bound to the RNA aptamer 130.

Further, as a modification of the detection unit 13, a configuration as shown in FIG.

In this modification, the RNA aptamer 130 of the detection unit 13 has a thiol group 131b in the terminal region 130c. In this case, it can be combined with gold through a gold-thiol bond. Therefore, it can be relatively easily fixed to another member via the gold bonded to the thiol group 131b. Specifically, as shown in FIG. 13 (b), the detection unit 13 includes a plurality of fixed films (Au) 13a positioned on the surface of the base 10 and a plurality of bonds respectively bonded to the fixed films 13a. The first substance 132 (the chain substance 13c (polyethylene glycol)) and the plurality of RNA aptamers (binding portions) 130 can be included.

Here, assuming that the first IDT electrode, the second IDT electrode, and the detection unit 13 arranged along the width direction of the sensor 100 are one set, the sensor 100 according to the present embodiment includes the set as shown in FIG. Are provided. Thereby, the detection object which reacts with one detection part 13 is set so that it may differ from the detection object which reacts with the other detection part 13, and two types of detection objects can be detected simultaneously by one sensor. It becomes possible. For example, an RNA aptamer 130 that can specifically bind to HbA1c may be provided in one detection unit 13, and an RNA aptamer that can specifically bind to HbA0 may be provided in the other detection unit 13. It is also possible to use aptamers and antibodies used for different detection targets as a set. In addition, for one of the two sets, a reference electrode may be provided instead of the detection unit 13 and used as a reference unit.

(Detection of detection object using detection element 3)
In order to detect the sample liquid in the detection element 3 using SAW, first, a predetermined voltage is applied to the first IDT electrode 11 from an external measuring instrument via the wiring 7 or the first extraction electrode 19. As a result, the surface of the substrate 10 is excited in the formation region of the first IDT electrode 11, and SAW having a predetermined frequency is generated. A part of the generated SAW propagates toward the detection unit 13, passes through the detection unit 13, and then reaches the second IDT electrode 12. In the detection unit 13, the RNA aptamer 130 of the detection unit 13 binds to the detection target (HbA1c) 135 in the sample liquid, and the weight of the detection unit 13 changes by the amount of the binding, so that the SAW that passes through the detection unit 13 changes. Characteristics such as phase change. When the SAW whose characteristics have changed in this way reaches the second IDT electrode, a voltage corresponding to the SAW is generated in the second IDT electrode. This voltage is output to the outside through the second extraction electrode 20, the wiring 7, etc., and by reading it with an external measuring instrument, the properties and components of the sample liquid can be examined.

Here, in order to guide the sample liquid to the detection unit 13, the sensor 100 uses a capillary phenomenon.

Specifically, as described above, the flow path 15 has an elongated tubular shape under the second cover member 2 by joining the second cover member 2 to the intermediate cover member 1A. Therefore, in consideration of the type of sample liquid, the material of the intermediate cover member 1A and the second cover member 2, the width or diameter of the flow channel 15 is set to a predetermined value, and the elongated tubular flow channel 15 is formed. Capillary action can occur. The width of the flow path 15 is, for example, 0.5 mm to 3 mm, and the depth is, for example, 0.1 mm to 0.5 mm. The flow path 15 includes an upstream portion 15a that is a portion from the inflow portion 14 to the front of the detection portion 13 and a downstream portion (extension portion) 15b that is a portion extending beyond the detection portion 13 beyond the detection portion 13. The second cover member 2 has an exhaust hole 18 connected to the extension 15b. When the sample liquid enters the flow path 15, the air present in the flow path 15 is released to the outside from the exhaust hole 18.

If a tube that causes such capillary action is formed by a cover member including the intermediate cover member 1A and the second cover member 2, the sample liquid flows through the flow path 15 by bringing the sample liquid into contact with the inflow portion 14. It is sucked into the cover member. Thus, since the sensor 100 itself includes a specimen liquid suction mechanism, the specimen liquid can be suctioned without using an instrument such as a pipette.

(Liquidity of flow path 15)
In the sensor 100 according to the present embodiment, the entire inner surface of the flow path 15 or a part of the inner surface, for example, the bottom surface and the wall surface of the flow path 15 are lyophilic. When the inner surface of the flow path 15 is lyophilic, capillary action is likely to occur, and the sample liquid is easily aspirated from the inflow portion 14.

For example, the lyophilic portion of the inner surface of the flow path 15 may have a contact angle with water of 60 ° or less. When the contact angle is 60 ° or less, capillary action is more likely to occur, and the sample liquid is more reliably aspirated into the flow path 15 when the sample liquid is brought into contact with the inflow portion. Hereinafter, it demonstrates in detail using Fig.3 (a). FIG. 3A is an enlarged sectional view showing a part of the sensor 100 of FIG.

In order to make the inner surface of the flow channel 15 lyophilic, for example, a method of applying hydrophilic treatment to the inner surface of the flow channel 15, a method of attaching a lyophilic film to the inner surface of the flow channel 15, and a flow A method of forming the cover member 2 constituting the path 15 with a lyophilic material can be employed. In particular, if a method of applying a hydrophilization treatment to the inner surface of the flow channel 15 and a method of attaching a lyophilic film to the inner surface of the flow channel 15, the sample liquid passes through the flow channel 15 along the lyophilic portion. Since it flows, it is possible to suppress the flow of the sample liquid to an unintended place and perform highly accurate measurement. In addition, according to these methods, even when a cover member made of a hydrophobic material is used, a capillary phenomenon can be caused, so that there is an advantage that the choice of materials that can be used as the cover member is increased.

As a method of applying a hydrophilic treatment to the inner surface of the flow path 15, for example, the inner surface of the flow path 15 is subjected to oxygen plasma to change the surface functional group by ashing, and then a silane coupling agent is applied, and finally polyethylene glycol is applied. May be applied. In addition, there is a method in which the inner surface of the flow path 15 is surface-treated using a treatment agent having phosphorylcholine.

In addition, in the method of attaching the lyophilic film, as the lyophilic film, a commercially available polyester film or polyethylene film that has been subjected to a hydrophilic treatment can be used. The lyophilic film may be formed only on the upper surface, the side surface, or the lower surface of the flow path 15 or a combination thereof.

(Positional relationship between the flow path 15 and the detection element 3)
In this embodiment, the flow path 15 for the sample liquid has a depth of about 0.3 mm, whereas the detection element 3 has a thickness of about 0.3 mm. As shown in FIG. The depth of 15 and the thickness of the detection element 3 are substantially equal. Therefore, if the detection element 3 is placed on the flow channel 15 as it is, the flow channel 15 is blocked. Therefore, in the sensor 100, as shown in FIG. 2B and FIG. 3, the element is composed of a first cover member 1 on which the detection element 3 is mounted and an intermediate cover member 1A joined on the first cover member 1. A housing recess 5 is provided. By accommodating the detection element 3 in the element accommodating recess 5, the flow path 15 for the sample liquid is prevented from being blocked. That is, the flow path 15 can be ensured by making the depth of the element accommodating recess 5 approximately the same as the thickness of the detecting element 3 and mounting the detecting element 3 in the element accommodating recess 5.

2B and FIG. 3, the height from the bottom surface of the element housing recess 5 to the upper surface of the substrate 10 is set to the depth of the element housing recess 5 from the viewpoint of sufficiently securing the sample liquid flow path 15. Or smaller (lower) than that. For example, if the height of the upper surface of the substrate 10 from the bottom surface of the element housing recess 5 is the same as the depth of the element housing recess 5, when the inside of the channel 15 is viewed from the inflow portion 14, The bottom surface and the detection unit 13 can be made substantially the same height.

The planar shape of the element receiving recess 5 may be, for example, a shape similar to the planar shape of the base 10, and the element receiving recess 5 may be set slightly larger than the base 10. More specifically, the element receiving recess 5 is large enough to form a gap of about 200 μm between the side surface of the base 10 and the inner wall of the element receiving recess 5 when the base 10 is mounted in the element receiving recess 5. That's it.

The detection element 3 is fixed to the bottom surface of the element housing recess 5 by a die bond material mainly composed of epoxy resin, polyimide resin, silicone resin or the like.

The end 19e of the first extraction electrode 19 and the wiring 7 are electrically connected by a thin metal wire 27 made of, for example, Au. The connection between the end 20e of the second extraction electrode 20 and the wiring 7 is the same. Note that the connection between the first extraction electrode 19 and the second extraction electrode 20 and the wiring 7 is not limited to the connection with the metal thin wire 27 but may be performed with a conductive adhesive such as Ag paste. Since a gap is provided in the connection portion between the first extraction electrode 19 and the second extraction electrode 20 and the wiring 7, when the second cover member 2 is bonded to the first cover member 1, Damage is suppressed. The 1st extraction electrode 19, the 2nd extraction electrode 20, the metal fine wire 27, and the wiring 7 are covered with the insulating sealing member. Since the first extraction electrode 19, the second extraction electrode 20, the fine metal wire 27 and the wiring 7 are covered with an insulating sealing member, it is possible to suppress corrosion of these electrodes and the like.

As described above, according to the sensor 100 according to the present embodiment, the flow path of the sample liquid extending from the inflow portion 14 to the detection portion 13 by accommodating the detection element 3 in the element accommodation recess 5 of the first cover member 1. 15 can be secured, and the sample liquid aspirated from the inflow portion by capillary action or the like can flow to the detection portion 13. That is, it is possible to provide the sensor 100 having the suction mechanism itself while using the thick detection element 3.

FIG. 6 is a plan view showing a manufacturing process of the sensor 100 of FIG.

First, as shown in FIG. 6A, a first cover member 1 on which terminals 6 and wirings 7 are formed is prepared.

Next, as shown in FIG. 6B, the intermediate cover member 1 </ b> A is laminated on the first cover member 1. Here, the intermediate cover member 1A includes a first upstream portion 1Aa and a first downstream portion 1Ab.

Next, as shown in FIG. 6 (c), the detection element 3 is mounted between the first upstream portion 1Aa and the first downstream portion 1Ab of the intermediate cover member 1A using the fine metal wires 27. Here, any of the steps of placing the intermediate cover member 1 </ b> A and the detection element 3 on the first cover member 1 may be executed first.

Next, as shown in FIG. 6D, the third substrate 2a of the second cover member 2 is laminated on the intermediate cover member 1A.

And as shown in FIG.6 (e), the sensor 100 which concerns on this embodiment is manufactured by laminating | stacking the 4th board | substrate 2b on the 3rd board | substrate 2a.

Next, a modified example of the sensor 100 according to the embodiment will be described.

<Modification>
7 is a plan view showing sensors 100a, 100b, and 100c according to a modification of the sensor 100 of FIG. 2, and FIGS. 7A and 7B are views corresponding to FIG. 6D, and FIG. These are figures corresponding to Fig.2 (a). In FIG. 7C, the vicinity of the detection element 3 is partially shown through the second cover member 2 for easy understanding of the structure.

The sensors 100a and 100b according to this modification are different from the sensor 100 according to the above-described embodiment in that the width of the intermediate cover member 1A and the second cover member 2 is larger than the width of the detection element 3. As shown in FIG. 7A, the sensor 100a is not provided with the intermediate cover member 1A (second downstream portion 1Ab) on the first cover member 1 downstream of the detection element 3. On the other hand, the sensor 100b is provided with an intermediate cover member 1A (second downstream portion 1Ab) on the first cover member 1 downstream of the detection element 3, as shown in FIG.

Next, the sensor 100c according to this modification is different from the sensor 100 according to the above-described embodiment in the arrangement of the terminals 6 with respect to the detection element 3.

Specifically, in the sensor 100, as shown in FIG. 2, the terminal 6 is arranged on the exhaust hole 18 side of the detection element 3 with respect to the end portion on the inflow portion 14 side. On the other hand, in the sensor 100c of this modification, as shown in FIG. 7C, at least a part of the terminal 6 is closer to the inflow portion 14 side than the end portion of the detection element 3 on the inflow portion 14 side. Is arranged.

Further, in the four terminals 6 arranged on one side of the detection element 3 with respect to the longitudinal direction of the flow path 15, the lengths of the wirings 7 connected to the two outer terminals 6 are substantially the same. The lengths of the wirings 7 connected to the two inner terminals 6 are substantially the same. According to this, it is possible to suppress the signal obtained by the detection element 3 from varying depending on the length of the wiring 7. Further, for example, one of the wirings 7 having substantially the same length is connected to a portion where the detection unit 13 of the detection element 3 detects the detection target, and the other wiring 7 having substantially the same length is connected to the detection element 3. If the detection unit is configured to be connected to the reference electrode for the detection target, it is possible to suppress the variation in the signal and to improve the reliability of detection.

8A and 8B are diagrams showing a sensor 101 according to a modification of the sensor 100 in FIG. 2, wherein FIG. 8A is a plan view, FIG. 8B is a sectional view in the length direction, and FIG. 8C is a sectional view in the width direction. is there. In FIG. 8A, the vicinity of the detection element 3 is partially shown through the second cover member 2 for easy understanding of the structure.

The sensor 101 according to this modification is different from the sensor 100 according to the above-described embodiment, and the first IDT electrode 11 and the second IDT electrode 12 are covered with an insulating member 28.

The insulating member 28 contributes to the oxidation prevention of the first IDT electrode 11 and the second IDT electrode 12. The insulating member 28 is made of, for example, silicon oxide, aluminum oxide, zinc oxide, titanium oxide, silicon nitride, or silicon. The thickness of the insulating member 28 is, for example, about 1/10 (10 to 30 nm) of the thickness of the first IDT electrode 11 and the second IDT electrode. The insulating member 28 may be formed over the entire top surface of the substrate 10 so as to expose the end 19e of the first extraction electrode 19 and the end 20e of the second extraction electrode 20.

Further, unlike the sensor 100 according to the above-described embodiment, the sensor 101 according to the present modification is provided with a filling member 9 in the gap between the detection element 3 and the intermediate cover member 1A.

The filling member 9 can include a material different from that of the intermediate cover member 1A and the base 10, and for example, a resin material such as PDMS can be used. The filling member 9 need not be provided in the entire region of the gap between the detection element 3 and the intermediate cover member 1 </ b> A, and may be provided only in a portion corresponding to the flow path 15, for example. Since the filling member 9 is positioned in the gap between the detection element 3 and the intermediate cover member 1A, it is possible to suppress the capillary phenomenon from being inhibited by the gap, and to aspirate the sample liquid more smoothly toward the detection element. Is possible.

FIG. 9 is a plan view showing a manufacturing process of the sensor 101 of FIG.

First, as shown in FIG. 9A, a first cover member 1 on which terminals 6 and wirings 7 are formed is prepared.

Next, as shown in FIG. 9B, the intermediate cover member 1 </ b> A is laminated on the first cover member 1. Here, the intermediate cover member 1A includes a first upstream portion 1Aa and a first downstream portion 1Ab.

Next, as shown in FIG. 9C, the detection element 3 is mounted between the first upstream portion 1Aa and the first downstream portion 1Ab of the intermediate cover member 1A using the fine metal wires 27. Here, any of the steps of placing the intermediate cover member 1 </ b> A and the detection element 3 on the first cover member 1 may be executed first.

Next, as shown in FIG. 9D, the filling member 9 is disposed in the gap between the detection element 3 and the intermediate cover member 1A.

Next, as shown in FIG. 9E, the third substrate 2a of the second cover member 2 is laminated on the intermediate cover member 1A.

And as shown in FIG.9 (f), the sensor 101 which concerns on this embodiment is manufactured by laminating | stacking the 4th board | substrate 2b on the 3rd board | substrate 2a.

FIG. 10 is a diagram illustrating a sensor 101a according to a modification of the sensor 100 in FIG. 2, and particularly illustrates a manufacturing process.

The sensor 101a according to the present modification is different from the sensor 100 according to the above-described embodiment in that the detection element 3 is surrounded by the intermediate cover member 1A in the top view. The filling member 9 is located in the gap between the detection element 3 and the intermediate cover member 1A so as to surround the outer periphery of the detection element 3, as shown in FIGS. According to this, since the step or the gap between the detection element 3 and its surroundings can be reduced in the flow path 15, the sample liquid can be smoothly flowed on the detection element 3. In addition, since the filling member 9 can cover a part of the wiring 7 and the conductive wire 27 that connects the detection element 3 and the wiring 7 in the region of the detection element 3 and the terminal 6, the contact between the filling member 9 and the sample liquid It is possible to suppress a decrease in detection sensitivity due to.

In this modification, the intermediate cover member 1A and the detection element 3 are placed on the first cover member 1 as shown in FIG. 10B, and then detected as shown in FIG. 10C. The element 3 and the wiring 7 are connected by a conducting wire 27. Instead, after the detection element 3 is placed on the first cover member 1 and the detection element 3 and the wiring 7 are connected by the conducting wire 27, the intermediate cover member 1A is placed on the first cover member 1. You may make it mount.

FIG. 11 is a plan view showing sensors 101b and 101c according to a modified example of the sensor 100 of FIG. 2, and corresponds to FIG. 6 (d).

The sensors 101b and 101c according to the present modification are different from the sensor 100 according to the above-described embodiment, and the filling member 9 includes a detection element 3 and an intermediate cover as shown in FIGS. 11 (a) and 11 (b). It is located along the longitudinal direction of the flow path 15 in the gap with the member 1A. According to this, since the level difference between the detection element 3 and both sides thereof can be reduced or the gap can be narrowed, the sample liquid can be smoothly flowed from the side to the detection element 3. In addition, since the filling member 9 can cover a part of the wiring 7 and the conductive wire 27 that connects the detection element 3 and the wiring 7 in the region of the detection element 3 and the terminal 6, the filling member 9 is in contact with the sample liquid. It is possible to suppress a decrease in detection sensitivity due to.

In addition, as shown in FIG. 11B, the detection member 3 is not only the gap between the detection element 3 and the intermediate cover member 1 </ b> A but also the detection element 3 among the conductive wires 27 for connecting the detection element 3 and the wiring 7. The part located on the upper surface of (base 10) can also be covered. According to this, it is possible to further suppress a decrease in detection sensitivity due to contact between the lead wire 27 and the sample liquid.

Hereinafter, examples of the RNA aptamer according to the above-described embodiment and a sensor using the RNA aptamer will be described.

(First embodiment)
Evaluation was performed using Biacore T200 (manufactured by GE Healthcare), which is an intermolecular interaction analyzer. This device has four flow cells.

SERIESSSensorChipSA (manufactured by GE Healthcare) was used as a substrate (base) for evaluation.

Hereinafter, a procedure for immobilizing various RNA aptamers on the Series SensorChip SA and detecting hemoglobin A1c will be described.

Here, the RNA aptamer obtained by transcription from a single-stranded DNA consisting of a complementary sequence of RNA (synthesized outside the company) was used. Further, ab98306 manufactured by Abcam Co. was used as the hemoglobin A1c specimen.

Further, as the running buffer, MgCl ₂ in HBS-P (manufactured by GE Healthcare) was used after adding MgCl ₂ as contained 1 mM (hereinafter referred to as _{HBS-P (+ MgCl 2)} .).

(1) All flow cells were washed with 10 mM NaOH at a flow rate of 20 μl / min for 1 minute.

(2) The above (1) was repeated three times.

(3) 5 μM TTTTTTTTTTTTTTTTTTT (SEQ ID NO: 24) having biotin at the 5 ′ end was flowed to one of the flow cells (hereinafter referred to as Fc2) at a flow rate of 5 μl / min for 6 minutes for immobilization. At this time, dilution was performed using an HBS-P (+ MgCl ₂ ) solution.

(4) Fc2 was immobilized with 5 μM TTTTTTTTTTTTTTTTTTT (SEQ ID NO: 24) having biotin at the 5 ′ end for 2 minutes at a flow rate of 5 μl / min.

(5) One of the flow cells (hereinafter referred to as Fc1) was immobilized by flowing 1 mg / ml biotin at a flow rate of 5 μl / min for 6 minutes. At this time, dilution was performed using an HBS-P (+ MgCl ₂ ) solution.

(6) The above flow cell was washed with 10 mM NaOH for 1 minute at a flow rate of 20 μl / min.

(7) The above (6) was repeated once.

(8) Fc2 was immobilized by flowing 0.5 μM RNA aptamer at a flow rate of 5 μl / min for 4 minutes, and then the dissociation state for 2 minutes was monitored. Here, as the RNA aptamer, AAAAAAAAAAAAAAAAA (SEQ ID NO: 23) was added to the 3 ′ end, and adenine and uracil in which the 2′-position was substituted with a fluorine group and annealed in advance were used. At this time, dilution was performed using an HBS-P (+ MgCl ₂ ) solution.

(9) A hemoglobin A1c sample diluted with HBS-P (+ MgCl ₂ ) so as to be 1 μM in the order of Fc1 and Fc2 is passed through the flow cells Fc1 and Fc2 for 4 minutes at a flow rate of 5 μl / min, and then dissociated for 2 minutes. The condition was monitored.

(10) The flow cells Fc1 and Fc2 were washed with 10 mM NaOH for 1 minute at a flow rate of 20 μl / min. With this operation, the RNA aptamer is removed, and the Series SensorChip SA returns to the state in which the above (7) is completed.

(11) After that, the above (8) to (10) are repeated, and sampleNo. 1 to 63 RNA aptamers (SEQ ID NOs: 25 to 87) were evaluated in order. The evaluation results are shown in Table 1.

Table 1 shows the amount of RNA aptamer immobilized, the amount of HbA1c detected, the number of RNA aptamer bases, and the binding ratio of HbA1c per RNA aptamer. When the value of the binding ratio of HbA1c per RNA aptamer is 0.1 or more, ◎, when 0.055 or more and less than 0.1, ○, and when greater than 0 and less than 0.055, Δ. , 0 or less was marked as x.

According to this, as shown in Table 1, the RNA aptamer having the base sequence represented by any of SEQ ID NOs: 25 to 47 showed high binding strength to HbA1c. In addition, the RNA aptamer having the base sequence represented by any of SEQ ID NOs: 25 to 39 showed particularly high binding strength to HbA1c.

(Second embodiment)
Table 2 shows the dissociation constants for HbA1c and HbA0 (non-glycated hemoglobin) for the major aptamers. The dissociation constant was obtained as follows.

Evaluation was performed using Biacore T200 (manufactured by GE Healthcare), which is an intermolecular interaction analyzer. This device has four flow cells.

SERIESSSensorChipSA (manufactured by GE Healthcare) was used as a substrate (base) for evaluation.

Here, the RNA aptamer used was a sequence consisting of only a common sequence from the sequences in Table 1 and 6 to 13 base pairs from both ends. This RNA was synthesized outside the company. In addition, as the hemoglobin A1c specimen, an HbA1c-certified practical standard substance (JCCRM423) (general incorporated association, manufactured by National Institute of Laboratory Medicine) was used. Further, as the hemoglobin A0 specimen, a substance obtained by removing HbA1c from the above-mentioned HbA1c certified practical reference material was used.

Further, as the running buffer, MgCl ₂ in HBS-P (manufactured by GE Healthcare) was used after adding MgCl ₂ as contained 1 mM (hereinafter referred to as _{HBS-P (+ MgCl 2)} .).

(1) All flow cells were washed with 10 mM NaOH at a flow rate of 20 μl / min for 1 minute.

(2) The above (1) was repeated three times.

(3) One of the flow cells (hereinafter referred to as Fc2) was immobilized by flowing 0.05 μM RNA aptamer having biotin at the 5 ′ end for 40 seconds at a flow rate of 20 μl / min. At this time, dilution was performed using an HBS-P (+ MgCl ₂ ) solution.

(4) One of the flow cells (hereinafter referred to as Fc1) was immobilized by flowing 1 mg / ml biotin at a flow rate of 5 μl / min for 6 minutes. At this time, dilution was performed using an HBS-P (+ MgCl ₂ ) solution.

(5) The flow cells Fc1 and Fc2 were washed with 10 mM NaOH for 1 minute at a flow rate of 20 μl / min.

(6) The above (5) was repeated three times.

(7) A diluted solution of the hemoglobin A1c sample was flowed in the order of Fc1 and Fc2 for 2 minutes at a flow rate of 50 μl / min, and then a running buffer was flowed for 2 minutes. At this time, dilution was performed using an HBS-P (+ MgCl ₂ ) solution.

(8) The flow cells Fc1 and Fc2 were washed with 10 mM NaOH for 1 minute at a flow rate of 20 μl / min.

(9) The specimen diluted so that the concentration of hemoglobin A1c was 0.01 μM was flowed in the order of Fc1 and Fc2 for 2 minutes at a flow rate of 50 μl / min, and then a running buffer was flowed for 2 minutes. At this time, dilution was performed using an HBS-P (+ MgCl ₂ ) solution.

(10) The flow cells Fc1 and Fc2 were washed with 10 mM NaOH for 1 minute at a flow rate of 20 μl / min.

(11) The above (9) and (10) were performed on the specimen diluted so that the hemoglobin A1c concentration was 0.02 μM, 0.05 μM, 0.1 μM, 0.2 μM, and 0.5 μM.

(12) Further, the above operations (7) to (11) were performed on the hemoglobin A0 specimen. However, the hemoglobin A0 concentration was 0 (diluted solution only), 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, and 5 μM.

(13) Thereafter, the BIAevaluation software was processed for the hemoglobin A1c and hemoglobin A0 concentration dependence of the SPR signal, and the dissociation constants KD_HbA1c and KD_HbA0 for the RNA aptamers hemoglobin A1c and hemoglobin A0 were determined. The results are shown in Table 2.

(Third embodiment: Example having biotin at the end)
Hereinafter, a procedure for immobilizing an RNA aptamer on a mounting substrate and detecting hemoglobin A1c will be described.

(1) A detection element 3 was prepared. Note that Au (gold) is used as the immobilization film 13a.

(2) The region including the detection unit 13 was washed with a piranha solution (mixed solution of concentrated sulfuric acid and 30% hydrogen peroxide).

(3) The region including the detection unit 13 was rinsed with water and ethanol.

In the following procedures (4) to (7), aptamers were immobilized on the immobilized film Au.

(4) 3,3′-dithiodipropionic acid was dropped onto the area containing the detection unit 13 and allowed to stand for 10 minutes, followed by washing with water.

(5) A liquid mixture of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide aqueous solution and N-hydroxysuccinimide aqueous solution is dropped on the region including the detection unit 13, left for 20 minutes, and then washed with water. The carboxyl group located at the end of 3,3′-dithiodipropionic acid was active esterified.

(6) A solution in which streptavidin is dissolved at a concentration of 0.1 mg / mL in a 1 mM HEPES buffer (pH 8.0) solution is dropped onto an area including the detection unit 13 and allowed to stand for 20 minutes. 0.0) solution was used for amide bond between the esterified carboxyl group and the amino residue in streptavidin.

(7) After replacing 1 mM HEPES buffer (pH 8.0) with HBS-P (+ MgCl ₂ ), HBS-P (+ MgCl ₂ ) so that the concentration of RNA aptamer having biotin at the 5 ′ end is 5 μM. The solution diluted in (1) was dropped on the area including the detection unit 13, left for 5 minutes, and then washed with HBS-P (+ MgCl ₂ ). Note that this process was not performed when the reference electrode was formed.

(8) A hemoglobin A1c sample diluted with HBS-P (+ MgCl ₂ ) so as to be 1 μM was introduced into the detection element 3 using the flow path 15, and the amount of phase change that occurred when reacted for 5 minutes was measured. .

According to this, the RNA aptamer could be immobilized with high stability and the HbA1c could be detected by using the binding of SA and biotin with high binding power.

(Example 4: Example having an amino group at the terminal)
Hereinafter, a procedure for immobilizing an RNA aptamer on a mounting substrate and detecting hemoglobin A1c will be described.

(1) A detection element 3 was prepared. Note that Au (gold) is used as the immobilization film 13a.

(2) The region including the detection unit 13 was washed with a piranha solution (mixed solution of concentrated sulfuric acid and 30% hydrogen peroxide).

(3) The region including the detection unit 13 was rinsed with water and ethanol.

In the following procedures (4) to (7), aptamers were immobilized on the immobilized film Au.

(4) Sensor element 3 is immersed in ethanol in which Carboxy-EG6-Undecanethiol (manufactured by Dojindo Laboratories) and Hydroxy-EG3-Undecanethiol (manufactured by Dojindo Laboratories) are dissolved in a desired concentration ratio for 16 hours. did.

(5) The detection element 3 was rinsed with ethanol and ultrapure water and dried with nitrogen.

(6) A mixed solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide aqueous solution, N-hydroxysuccinimide aqueous solution, and hydrochloric acid was dropped on the region including the detection unit 13 and left for 10 minutes.

(7) The mixed solution was removed, and a mixed solution having the same composition as that described above was dropped onto the region including the detection unit 13 and left for 10 minutes.

(8) The above (7) was repeated four times.

(9) The carboxyl group located at the end of Carboxy-EG6-Undecanethiol was active esterified by washing with 10 mM sodium acetate aqueous solution (pH 5.0).

(10) A region containing a detection unit 13 by dissolving an RNA aptamer having an amino group at the 5 ′ end in a 100 mM HEPES buffer (pH 7.4) solution containing 4.5 M NaCl so as to have a desired concentration. And then left for 30 minutes.

(11) The above solution was removed, and an RNA aptamer solution having the same concentration as that described above was dropped onto the region including the detection unit 13 and allowed to stand for 30 minutes.

(12) After repeating the above (11), the region containing the detection unit 13 is washed with 10 mM NaOH and HBS-P (+ MgCl ₂ ) to be present at the 5 ′ end of the esterified carboxyl group and the RNA aptamer. The amino group to be amide-bonded. When forming the reference electrode, the operations (10) and (11) were not performed.

(13) A hemoglobin A1c sample diluted with HBS-P (+ MgCl ₂ ) so as to be 5 μM was introduced into the detection element 3 using the flow path 15 and the amount of phase change generated when reacted for 30 minutes was measured. .

According to this, by using an amide bond having high stability, the RNA aptamer could be immobilized with high stability and HbA1c could be detected.

(Fifth embodiment: verification of core sequence)
Below, the result evaluated about the influence of parts other than a common arrangement | sequence is shown.

Evaluation was performed using Biacore T200 (manufactured by GE Healthcare), which is an intermolecular interaction analyzer. This device has four flow cells.

SERIESSSensorChipSA (manufactured by GE Healthcare) was used as a substrate (base) for evaluation.

Here, the RNA aptamer is the No. in Table 2. For 64 (SEQ ID NO: 88), all base pairs other than the common sequence are GC pairs and the base pair chain length is changed. These RNAs are synthesized outside the company. When the secondary structure of the RNA aptamer whose base pair chain length is changed is displayed using the software for predicting the secondary structure in the same manner as described above, the structure shown in FIG. In addition, as the hemoglobin A1c specimen, an HbA1c-certified practical standard substance (JCCRM423) (general incorporated association, manufactured by National Institute of Laboratory Medicine) was used.

Further, as the running buffer, MgCl ₂ in HBS-P (manufactured by GE Healthcare) was used after adding MgCl ₂ as contained 1 mM (hereinafter referred to as _{HBS-P (+ MgCl 2)} .).

(1) All flow cells were washed with 10 mM NaOH at a flow rate of 20 μl / min for 1 minute.

(2) The above (1) was repeated three times.

(3) 2 μM RNA aptamer having biotin at the 5 ′ end was immobilized on one of the flow cells (hereinafter referred to as Fc2) at a flow rate of 5 μl / min for 6 minutes. At this time, dilution was performed using an HBS-P (+ MgCl ₂ ) solution.

(4) Fc2 was immobilized with 2 μM RNA aptamer having biotin at the 5 ′ end for 2 minutes at a flow rate of 5 μl / min. At this time, dilution was performed using an HBS-P (+ MgCl ₂ ) solution. FIG. 14B shows the signal intensity when the signal is fixed. It can be seen that each aptamer is immobilized in substantially the same amount.

(5) The flow cells Fc1 and Fc2 were washed with 10 mM NaOH for 1 minute at a flow rate of 20 μl / min.

(6) The above (5) was repeated three times.

(7) The hemoglobin A1c specimen diluted to a concentration of 5 μM was flowed in the order of Fc1 and Fc4 at a flow rate of 5 μl / min for 4 minutes, and then a running buffer was flowed for 2 minutes. At this time, dilution was performed using an HBS-P (+ MgCl ₂ ) solution.

The evaluation result is shown in FIG. It can be seen that hemoglobin A1c can be bound even if the base pair portion is changed to only the GC pair. That is, it can be seen that the RNA aptamer binds to hemoglobin A1c by the common sequence and the base pair in Table 1. Furthermore, it can be seen that the longer the base pair chain length, the higher the binding performance with hemoglobin A1c.

The present invention is not limited to the above embodiment, and may be implemented in various modes.

For example, in the above-described embodiment, the detection unit 13 has been described as being composed of a metal film and an RNA aptamer 130 immobilized on the surface of the metal film. The RNA aptamer 130 may be immobilized on the surface.

In the embodiment described above, the detection element 3 is made of a surface acoustic wave element. However, the detection element 3 is not limited to this, and for example, a detection in which an optical waveguide is formed so that surface plasmon resonance occurs. Element 3 may be used. In this case, for example, a change in the refractive index of light in the detection unit is measured. In addition, the detection element 3 in which a vibrator is formed on a piezoelectric substrate such as quartz can be used. In this case, for example, a change in the oscillation frequency of the vibrator is measured.

Also, as the detection element 3, a plurality of types of devices may be mixed on a single substrate. For example, an enzyme electrode method enzyme electrode may be provided next to the SAW element. In this case, in addition to the immunization method using an antibody or RNA aptamer, measurement by an enzyme method is possible, and the number of items that can be examined at a time can be increased.

In the above-described embodiment, an example in which one detection element 3 is provided has been described. However, a plurality of detection elements 3 may be provided. In this case, the element accommodating recess 5 may be provided for each detection element 3 or the element accommodating recess 5 having a length or width that can accommodate all the detection elements 3 may be formed.

In the above-described embodiment, the example in which the first cover member 1, the intermediate cover member 1A, and the second cover member 2 are separate members has been described. You may use what was done.

DESCRIPTION OF SYMBOLS 1 ... 1st cover member 1A ... Intermediate | middle cover member 1Aa ... 1st upstream part 1Ab ... 1st downstream part 2 ... 2nd cover member 2a ... 3rd board | substrate 2b ... 4th board | substrate 3 ... detection element 4 ... recessed part formation site 5 ... element accommodating recessed part 6 ... terminal 7 ... wiring 9 ... filling member 10 ... base | substrate 11 ... 1st IDT Electrode 12 ... 2nd IDT electrode 13 ... Detection part 13a ... Immobilization film | membrane (immobilization member)
13c ... chain substance 130 ... RNA aptamer (binding part)
130a ... common sequence region 130b ... stem region 130b1 ... first nucleotide sequence region 130b2 ... second nucleotide sequence region 130c ... end region 130c1, ... 3 'end 130c2, ... 5' Terminal 131a ... Biotin 131b ... thiol group 131c ... amino group 132 ... first substance 133 ... double strand 134 ... streptavidin 135 ... detection target (hemoglobin A1c)
14 ... Inflow part 15 ... Flow path 15a ... Upstream part 15b ... Downstream part (extension part)
18 ... exhaust hole 19 ... first extraction electrode 19e ... end 20 ... second extraction electrode 20e ... end 27 ... conducting wire (fine metal wire)
28 ... Insulating member 100, 101 ... Sensor

Claims (22)

1. AGNNNNGWGWSC (SEQ ID NO: 1) (excluding nucleotide sequences in which the 3rd to 5th N from the 5 ′ end are adenine, guanine, and guanine, respectively),
   AGNNNNWGGWSC (SEQ ID NO: 2),
   AGNNNNNWGGWSC (SEQ ID NO: 3) or AUGNNNNAGACC (SEQ ID NO: 4) contains a consensus sequence (N is adenine, guanine, cytosine or uracil, W is adenine or uracil, and S is guanine or cytosine) And an RNA aptamer having binding ability to HbA1c.
2. The consensus sequence is
   AGNHNWGGWSC (SEQ ID NO: 5),
   AGNHNNWGGWSC (SEQ ID NO: 6),
   AGNHNNNNGWGWSC (SEQ ID NO: 7), or AUGNHNAGGACC (SEQ ID NO: 8), the base sequence (N is adenine, guanine, cytosine or uracil, H is adenine, cytosine or uracil, W is adenine or uracil, The RNA aptamer according to claim 1, wherein S is guanine or cytosine).
3. The consensus sequence is
   AGNNNNWGGWSC (SEQ ID NO: 1) (excluding nucleotide sequences in which 3rd to 5th N from the 5 ′ end are adenine, guanine and guanine, respectively)
   AGNNNNNGWGWSC (SEQ ID NO: 2) or the base sequence shown by AGNNNNNNGWGWSC (SEQ ID NO: 3) (N is adenine, guanine, cytosine or uracil, W is adenine or uracil, and S is guanine or cytosine) The RNA aptamer according to claim 1.
4. The consensus sequence is
   A nucleotide sequence represented by AGNNNAGGACC (SEQ ID NO: 9) (N is adenine, guanine, cytosine, or uracil, and the third to fifth Ns counted from the 5 ′ end are adenine, guanine, and guanine, respectively. The RNA aptamer according to claim 1, wherein
5. The consensus sequence is
   The RNA aptamer according to claim 4, which is a base sequence represented by AGWYDAGGACC (SEQ ID NO: 10) (W is adenine or uracil, Y is uracil or cytosine, and D is adenine, guanine or uracil).
6. The consensus sequence is
   AGACGAGGACC (SEQ ID NO: 11),
   AGAUUAGGACC (SEQ ID NO: 12),
   AGACAAGGACC (SEQ ID NO: 13),
   AGAUGAGGACC (SEQ ID NO: 14),
   AGUCGAGGACC (SEQ ID NO: 15),
   AGACUAGGACC (SEQ ID NO: 16),
   AGAUUGGAGGAGC (SEQ ID NO: 17),
   AGACGAGGUGC (SEQ ID NO: 18),
   AGGAAUGGACC (SEQ ID NO: 19),
   AGAUUGAGGACC (SEQ ID NO: 20),
   The RNA aptamer according to claim 1, which is a base sequence represented by AGAACGAGGACC (SEQ ID NO: 21) or AUGAUUAGGACC (SEQ ID NO: 22).
7. The consensus sequence is
   AGACGAGGACC (SEQ ID NO: 11),
   AGAUUAGGACC (SEQ ID NO: 12),
   AGACAAGGACC (SEQ ID NO: 13),
   AGAUGAGGACC (SEQ ID NO: 14),
   The RNA aptamer according to claim 6, which is a base sequence represented by AGUCGAGGACC (SEQ ID NO: 15) or AGACUAGGACC (SEQ ID NO: 16).
8. One of guanine existing from the first to the third position from the 5 ′ end of the common sequence and one of cytosine existing from the first position to the third position from the 3 ′ end of the common sequence. The RNA aptamer according to any one of claims 1 to 7, which forms a base pair.
9. The RNA aptamer according to any one of claims 1 to 8, further comprising a first nucleotide sequence that binds to the 5 'end of the common sequence and a second nucleotide sequence that binds to the 3' end of the common sequence. .
10. The aptamer according to claim 9, wherein the first nucleotide sequence includes at least one nucleotide that forms a base pair with the nucleotide in the second nucleotide sequence.
11. The RNA aptamer according to claim 9, wherein the first nucleotide sequence or the second nucleotide sequence includes at least one nucleotide that forms a base pair with the nucleotide in the common sequence.
12. The RNA aptamer according to claim 10 or 11, wherein the common sequence, the first nucleotide sequence, and the second nucleotide sequence can form a stem-loop structure.
13. The RNA aptamer according to claim 12, wherein the number of base pairs in the stem-loop structure is at least 6 or more.
14. The RNA aptamer according to any one of claims 10 to 13, wherein at least one of the base pairs is a base pair of cytosine and guanine.
15. The RNA aptamer according to any one of claims 1 to 14, wherein at least one nucleotide in the common sequence has a fluorine group at the 2'-position of ribose.
16. The RNA aptamer according to any one of claims 1 to 15, wherein the total number of nucleotides is 23 to 96.
17. 2. The RNA aptamer according to claim 1, comprising a base sequence represented by any of SEQ ID NOs: 25 to 47 and 88 to 100.
18. The RNA aptamer according to any one of claims 1 to 17, further comprising biotin located at a terminal.
19. The RNA aptamer according to claim 18, further comprising streptavidin bound to the biotin.
20. The RNA aptamer according to any one of claims 1 to 17, further comprising an amino group located at a terminal.
21. A substrate;
    The RNA aptamer according to any one of claims 1 to 19, which is immobilized on the substrate.
22. The sensor according to claim 21, further comprising an immobilization member located between the RNA aptamer and the substrate and bonded to at least one of the RNA aptamer and the substrate.

Images