JP-7855468-B2 - Chemical sensors using chain exchange reactions

JP7855468B2JP 7855468 B2JP7855468 B2JP 7855468B2JP-7855468-B2

Inventors

杉崎吉昭
三木弘子

Assignees

株式会社東芝

Dates

Publication Date: 20260508
Application Date: 20220916

Claims (18)

Nucleic acid probes for capturing target substances, A sensor element with the nucleic acid probe immobilized on its surface, A chemical sensor comprising a liquid film covering the sensor element, The nucleic acid probe is a double-stranded nucleic acid consisting of a first nucleic acid and a second nucleic acid bound to the first nucleic acid. The first nucleic acid is a single-stranded nucleic acid comprising a first base sequence, a second base sequence consisting of a base sequence complementary to the first base sequence, and a third base sequence having one end bound to the first base sequence and the other end bound to the second base sequence. The second nucleic acid is a single-stranded nucleic acid that includes a fourth base sequence which is complementary to a part of the second base sequence of the first nucleic acid. The base sequence constituting the binding site that captures the target substance is included in the third base sequence. When the third base sequence captures the target substance, the first nucleic acid and the second nucleic acid dissociate, and a secondary structure is formed within the first nucleic acid having a stem portion formed by the pairing of the first base sequence and the second base sequence, and a loop portion or bulge structure containing the third base sequence. The base length of the fourth base sequence is shorter than the base length of the second base sequence. Chemical sensor.
The chemical sensor according to claim 1, wherein the fourth base sequence and the third base sequence do not have complementary strands to each other.
The chemical sensor according to claim 1, wherein the first nucleic acid of the nucleic acid probe includes a spacer sequence of one, two, or three bases between the base sequence constituting the binding site of the third base sequence and the second base sequence.
The chemical sensor according to claim 1, comprising a single-base spacer between the base sequence constituting the binding site of the third base sequence and the first base sequence.
The chemical sensor according to claim 1, wherein the first nucleic acid, dissociated from the second nucleic acid by the capture of the target substance by the third base sequence, forms a higher-order structure.
The chemical sensor according to claim 1, wherein the secondary structure of the first nucleic acid, formed by the capture of the target substance by the third base sequence, is a hairpin loop, an internal loop, a bulge, a pseudoknot, a three-way junction, or a four-way junction.
The chemical sensor according to claim 1, wherein the sensor element is one of the following: graphene FET, ISFET, SPR, and QCM.
The chemical sensor according to claim 1, wherein the nucleic acid probe is immobilized on the surface of the sensor element by immobilizing either the first nucleic acid or the second nucleic acid.
The chemical sensor according to claim 1, wherein the fourth base sequence has a base sequence that is a mismatch with the second base sequence.
The fourth base sequence of the second nucleic acid further includes a base sequence that does not pair with the second base sequence, or the second base sequence includes a base sequence that does not pair with the fourth base sequence. The chemical sensor according to claim 1, comprising a nucleic acid probe including a double-stranded bulge structure formed by the bonding of the second base sequence of the first nucleic acid and the fourth base sequence of the second nucleic acid.
The chemical sensor according to claim 1, wherein the second nucleic acid is PNA, in which the fourth base sequence and the amino acid sequence of a polycation are bonded.
The chemical sensor according to claim 1, wherein the liquid film contains a polycationic amino acid or PNA having a polycationic amino acid sequence.
The amino acid sequence of the polycation is part of a PNA that includes a base sequence complementary to the base sequence of a part of the second nucleic acid. The polycation binds to a portion of the base sequence of the second nucleic acid to form a double helix with the second nucleic acid. The chemical sensor according to claim 12 .
A donor fluorescent dye and an acceptor are bound to any two of the first base sequence, the second base sequence, and the third base sequence, respectively. Fluorescence resonance energy transfer (FRET) occurs between the base sequence to which the donor fluorescent dye is bound and the base sequence to which the acceptor is bound. The chemical sensor according to claim 1.
A method for detecting a target substance using the chemical sensor described in claim 1, (S1) Prepare the chemical sensor described in claim 1 and a sample containing the target substance; (S2) Bringing the sample into contact with the liquid film of the chemical sensor; and, (S3) Detecting the DNA strand exchange reaction that occurs when the target substance is captured by the nucleic acid probe; The detection method, including the method described above.
A method for manufacturing the chemical sensor described in claim 1, (S1) A sensor element having the first nucleic acid immobilized on its surface and coated with the liquid film, wherein the first nucleic acid includes a stem portion formed by the bonding of the first base sequence and the second base sequence, and a solution containing the second nucleic acid; (S2) Denature the first nucleic acid that is immobilized on the surface of the sensor element prepared in (S1) so as to dissociate the bond between the first base sequence and the second base sequence; (S3) Dropping the solution containing the second nucleic acid onto the surface of the sensor element; and (S4) Combining the first nucleic acid and the second nucleic acid to form the nucleic acid probe; Methods that include...
The sensor element prepared in (S1) further comprises a heating device for heating the solution constituting the liquid film and a cooling device for cooling the solution constituting the liquid film. The above (S2) is carried out by heating the solution constituting the liquid film with the heating device to denature the first nucleic acid. The method according to claim 16 , wherein (S4) is performed by cooling the solution constituting the liquid film with the cooling device.
The chemical sensor further comprises a device for supplying a low-salt concentration solution to the surface of the sensor element, and a device for supplying a high-salt concentration solution to the surface of the sensor element. After (S1) and before (S2), replace the solution constituting the liquid film with the low-salt concentration solution supplied from the apparatus for supplying the low-salt concentration solution to the sensor element surface; and after (S4), replace the solution constituting the liquid film with the high-salt concentration solution supplied from the apparatus for supplying the high-salt concentration solution to the sensor element surface; The method according to claim 16, including the method described in claim 16 .

Description

Embodiments of the present invention relate to a chemical sensor using a chain exchange reaction. There is a need for chemical sensors equipped with nucleic acid probes that can measure with high sensitivity. Figure 1 is a cross-sectional view showing an example of a chemical sensor according to the first embodiment.Figure 2(a) is a schematic diagram showing an example of a nucleic acid probe of the chemical sensor of the first embodiment, (b) is a diagram showing the progress of the strand exchange reaction in the nucleic acid probe, and (c) is a diagram showing the nucleic acid probe after the strand exchange reaction has been completed and a stable higher-order structure has been formed.Figure 3 is an energy level diagram for the nucleic acid probe provided in the chemical sensor of the first embodiment, showing the change in free energy when the nucleic acid probe forms a double helix and when it forms a single helix.Figure 4(a) shows an example of a second nucleic acid containing a polycation, which is provided in the chemical sensor of the second embodiment, and (b) is a schematic diagram showing how the second nucleic acid containing a polycation and other base sequences assemble in a strand exchange reaction.Figure 5 is an energy level diagram for the nucleic acid probe provided in the chemical sensor of the second embodiment, showing the change in free energy due to the capture of the target substance.Figure 6 is a cross-sectional view showing an example of a chemical sensor according to the third embodiment.Figure 7(a) shows an example of a polycation provided by the chemical sensor of the second embodiment, and (b) is a schematic diagram showing how the polycation and other base sequences assemble in a chain exchange reaction.Figure 8 is a flowchart of a fourth embodiment, which describes an analysis method using a chemical sensor.Figure 9 is a flowchart relating to a fifth embodiment, which is a method for manufacturing a chemical sensor.Figure 10 is a graph showing the time-dependent change in the drain current flowing through the sensor element when the concentration of the target substance is changed in stages, as measured in Example 1.Figure 11 shows the gate voltage dependence (IdVg characteristic) of the drain current of the FET sensor equipped with a graphene film for each measurement period, as measured in Example 1. The embodiments will be described below with reference to the attached drawings. Note that in each embodiment, substantially identical components are denoted by the same reference numerals, and their descriptions may be partially omitted. The drawings are schematic, and the relationship between the thickness of each part and its planar dimensions, the ratio of the thicknesses of each part, etc., may differ from those of reality. (First Embodiment)・Chemical Sensor According to the first embodiment, a chemical sensor (hereinafter referred to as "sensor 1") is provided that includes a nucleic acid probe that detects a target substance by undergoing a chain exchange reaction. Sensor 1 comprises a sensor element 2 and a liquid film 3 arranged to cover the sensor element 2, with a nucleic acid probe 4 immobilized on the surface of the sensor element 2. Any type of sensor element may be selected for sensor element 2, as long as it is configured to sense changes in the secondary structure of the nucleic acid probe 4, which will be described later. Sensor element 2 may be, for example, a graphene field-effect transistor (GFET), an ion-sensitive field-effect transistor (ISFET), a surface plasmon resonance element (SPR), or a quartz crystal microbalance (QCM). For example, if sensor 1 is a type of FET sensor, as shown in Figure 1, the sensor element 2 is arranged such that the gate electrode 5 makes contact via a liquid film 3, and a source electrode 6 is electrically connected to one end and a drain electrode 7 to the other end. A circuit for applying voltage (i.e., gate voltage) is connected to the gate electrode 5. A circuit for applying voltage is also formed between the source electrode 6 and the drain electrode 7, and an ammeter (not shown) for measuring the drain current flowing through this circuit is positioned there. The source electrode 6 and drain electrode 7 may be covered with an insulating protective film 8. The liquid film 3 is positioned so that its surface 3a is in contact with the sample containing the target substance, and it covers the sensor element 2, immersing the immobilized nucleic acid probe 4 on the surface 2a of the sensor element 2. The liquid film 3 is composed of a measurement solution capable of dissolving the target substance. As the solvent for the measurement solution, water can be selected, for example, and the solute may include any reagents necessary for the measurement or storage of the sensor 1 (e.g., stabilizers, pH adjusters, ions, etc.). The nucleic acid probe 4 is a double-stranded nucleic acid consisting of a first nucleic acid and a second