JP2021099293A - Interaction detection method, interaction detection device, and biochip regeneration kit - Google Patents

Abstract

To provide an interaction detection method capable of returning a biochip to its initial state even if a ligand is metal ion demanding while shortening time to start next analysis as much as possible.SOLUTION: An interaction detection method includes: a detection step of detecting interaction by bringing a sample containing analyte into contact with a ligand; and a regeneration step of returning the ligand to a state before bringing the same into contact with the sample. In the interaction detection method, the ligand requires a metal ion to bind to the analyte, and in the regeneration process, a solution for removing the analyte from the ligand and supplying the metal ion to the ligand, is used.SELECTED DRAWING: Figure 3

Description

The present invention relates to an interaction detection method and the like.

As a method of detecting the interaction between the ligand and the target substance (analyte) contained in the sample, using a biochip in which a biomolecule such as a protein serving as a binding molecule (ligand) is immobilized on the surface of the substrate. For example, as described in Patent Document 1, there is a method using a surface plasmon resonance phenomenon.

In such an interaction detection method, in order to use the biochip many times and repeatedly and to accurately detect the interaction with the analytes, the ana that is bound to the ligand before each supply of the analytes. It is necessary to remove the light and return the biochip to its initial state.

WO2019 / 044445A1

Therefore, in order to remove the analyte from the ligand, as described in Patent Document 1, when the announcer was removed from the ligand using phosphate buffered saline (PBS), this operation alone was sufficient for biochip. The inventor has noticed that the chip may not return to its initial state.
The reason for this result is that, for example, when the ligand requires a metal ion for binding to the analyte, the metal ion also dissociates when the ligand and the analyte dissociate. , It is possible that the next supplied analyze cannot be combined.
In such a case, as a method of returning the biochip to the initial state, it is conceivable to add a step of supplying metal ions to the ligand after the washing step.
However, if a step of supplying metal ions is added, there is a problem that the time until the next analysis is started becomes long.
The present invention has been made in view of such a problem, and even when the ligand is a metal ion-requiring one, until the biochip is returned to the initial state and the next analysis is started. The main purpose is to provide an interaction detection method capable of shortening the time required for.

That is, the interaction detection method according to the present invention includes a detection step of contacting a sample containing an analyte with a ligand to detect the interaction, and a regeneration step of returning the ligand to a state before contact with the sample. An interaction detection method in which the ligand requires a metal ion for binding to an analyte, and in the regeneration step, the allate is removed from the ligand and the metal ion is attached to the ligand. It is characterized by using a solution for supplying.

According to such an interaction detection method, in the regeneration step, the solution used has both a function of removing the analyte from the ligand and a function of supplying metal ions to the ligand, so that the ligand requires metal ions. Even if it is, the time until the biochip is returned to the initial state and the next analysis is started can be shortened as much as possible.

The solution contains a salt of the metal ion, and the concentration of the salt is preferably 1 mol / L or more and 5 mol / L or less.

It is preferable that the metal ion contains a divalent metal ion, for example, magnesium ion or calcium ion.

Specific embodiments of the present invention include those in which the ligand contains any one or more of lectins, antibodies and cell adhesion molecules.

It is preferable that the analyst contains an exosome.

As a specific embodiment of the interaction detection method according to the present invention, a method using a surface plasmon resonance phenomenon can be mentioned.

A biochip in which a ligand is fixed on the surface of a base material, a flow path for accommodating the biochip inside, a distribution mechanism for distributing a sample solution containing an analyzer in the flow path, and a ligand on the surface of the biochip. An interaction detection device comprising a detection unit 4 for detecting the interaction between the ligand and the analyte, wherein the ligand requires a metal ion for binding to the analyte, and the distribution mechanism comprises. An interaction detection device characterized in that after the sample solution is circulated, a solution for removing the allite from the ligand and supplying the metal ion to the ligand is circulated in the flow path. Is also an embodiment of the present invention.

A biochip in which a ligand that requires a metal ion for binding to an exosome, which is an analite, is immobilized on the surface of a substrate, and a solution that removes the allate from the ligand and supplies the metal ion to the ligand. A biochip regeneration kit comprising the above is also an embodiment of the present invention.

According to the present invention, even when the ligand is metal ion-requiring, the time until the biochip is returned to the initial state and the next analysis is started can be shortened as much as possible.

The schematic diagram which shows the whole structure of the interaction detection apparatus which concerns on one Embodiment of this invention. The schematic diagram explaining the interaction mechanism on the biochip which concerns on this embodiment. The flowchart which shows the procedure of the interaction detection method which concerns on one Embodiment of this invention. The graph which shows the result of the interaction detection method which concerns on Example of this invention. The photograph which shows the result of the interaction detection method which concerns on this Example.

Hereinafter, an embodiment of the present invention will be described with reference to the drawings.
The interaction detection device 100 according to the present embodiment uses, for example, as shown in FIG. 1, a biochip 1 in which a binding molecule B (ligand B) is immobilized on the surface thereof, and the ligand B and the target substance A ( This is a microarray type SPRi device that detects the interaction with Analite A) using, for example, a surface plasmon resonance phenomenon.
In this embodiment, an exosome surface molecule existing on the surface of an exosome is used as an analyze A to detect an interaction between a ligand B and an exosome surface molecule.

Exosomes are extracellular vesicles with a diameter of about 50 to 150 nm covered with a phospholipid bilayer membrane. This exosome retains the same molecules (proteins, RNAs, lipids, etc.) as cells that secrete exosomes on the surface of the exosome and within the exosome. Therefore, if the molecule retained by the exosome can be detected as a tumor marker, it can be established as a new diagnostic method for tumors.
The cells that secrete exosomes are not particularly limited, such as animal cells, plant cells, and microbial cells. Animal cells include mammalian cells, and the mammalian cells are not limited to the following, but are, for example, erythrocytes, hepatocytes, splenocytes, nerve cells, glial cells, pancreatic β cells, and bone marrow cells. , Mesangium cells, Langerhans cells, epidermal cells, epithelial cells, cup cells, endothelial cells, smooth muscle cells, fibroblasts, fibrous cells, muscle cells, fat cells, immune cells (eg macrophages, T cells, B cells, natural) Killer cells, obese cells, neutrophils, basal spheres, eosinophils, monospheres), macronuclear cells, synovial cells, cartilage cells, bone cells, osteoblasts, osteoclasty cells, mammary gland cells or stromal cells, Alternatively, precursor cells, stem cells, cancer cells, cultured cells and the like of these cells can be mentioned.

The interaction detection device 100 includes the biochip 1, a flow path 2 for accommodating the biochip 1, a flow mechanism 3 for circulating a sample solution containing ananalite A in the flow path 2, and the above-mentioned. A detection unit 4 that detects the interaction between the ligand B and Anna Reed A on the surface of the biochip 1, a calculation unit 5 that calculates the strength of the interaction based on the signal from the detection unit 4, and the calculation unit. It is provided with a display unit 6 for displaying the result calculated by.

The biochip 1 is, for example, a prism (not shown) attached to a surface of the base material 11 and a ligand B fixed to the surface thereof, and a surface of the base material 11 opposite to the surface on which the ligand B is fixed. It is equipped with.
As the base material 11, various materials such as a film made of a synthetic resin such as polystyrene, polyacrylamide, and silicon, glass, a metal thin film, or a nitrocellulose film can be used. In this embodiment, a metal thin film is used as the base material 11.
Examples of the metal constituting the metal thin film include gold, silver, copper, and aluminum.
It is preferable that a succinimide-activated carboxy group is immobilized on the surface of the base material 11 on the side on which the ligand B is immobilized.
The size and shape of the base material 11 can be appropriately changed depending on the analyzer used, but in the present embodiment, a rectangular plate having a width of about 12 mm and a length of about 23 mm is adopted.
The prism has a triangular shape in the present embodiment, but is not particularly limited, and examples thereof include a trapezoidal prism, a circular prism, and a semi-cylindrical prism. It is preferable to use a prism having a refractive index of, for example, 1.5 to 1.8.

The ligand B is, for example, a protein derived from a living body, and in the present embodiment, it is bound to a carboxy group immobilized on the surface of the base material 11.
The solid phase of the ligand B can be carried out by spotting a ligand solution prepared by adjusting the ligand B to an appropriate concentration with a buffer solution or the like on the base material 11 and allowing it to stand. The concentration of the ligand solution can be appropriately changed depending on the amount of ligand B to be immobilized, but is preferably about 1 mg / ml, for example. The standing time can be changed as appropriate, but it is preferably about 8 to 16 hours, for example.

The flow path 2 includes, for example, a tube 21 made of vinyl chloride or the like and having an inner diameter of about 400 μm, and a flow cell 22 made of vinyl chloride or the like connected to the tube 21. The flow cell 22 houses the biochip 1 inside, and a liquid containing a sample flows between the surface of the biochip 1 on the side where the ligand B is immobilized and the inner wall of the flow cell. It is configured to form a gap for the purpose.

The distribution mechanism 3 includes, for example, a sample introduction port 31, a solution bottle 32, and a pump 33.
The sample introduction port 31 opens with respect to the flow path 2, for example.
The solution bottle 32 is for storing various solutions flowing in the flow path 2, and a required number of bottles are prepared for each solution to be used. Each of these solution bottles 32 is connected to a tube constituting the flow path 2.
The pump 33 is arranged on the flow path 2, for example, and allows various solutions to flow through the flow path 2.

The detection unit 4 is, for example, a light source (not shown) that irradiates the surface of the biochip 1 with light, and a sensor (not shown) that detects the reflected light associated with the SPR phenomenon induced by the binding of exosomes to the surface of the biochip 1. It is equipped with.

The calculation unit calculates and outputs the amount of change in the reflected light detected by the detection unit 4 as the reflectance (%).
In this embodiment, the information processing circuit plays a role in this calculation unit.
This information processing circuit includes a digital circuit composed of a CPU, a memory, a communication port, etc., an analog circuit including a buffer, an amplifier, etc., and an AD converter, a DA converter, etc. that mediate between these digital circuits and the analog circuit. It was done. Then, when the CPU and its peripheral devices cooperate according to a predetermined program stored in the memory, this information processing circuit exerts a function as the calculation unit.

The display unit outputs the change in reflectance calculated by the calculation unit as a color tone image.

Thus, in the present embodiment, a ligand B that requires a metal ion C for binding to the analyte A is used. The ligand B contains, for example, any one of lectins, antibodies, cell-cell adhesion factors and the like.

The lectin may be, for example, a sugar-binding or sugar-chain-binding protein or glycoprotein having the property of aggregating complex sugars such as cells or glycoproteins and glycolipids other than antibodies, and is particularly limited. Not done.
Examples of the lectin include animal lectins, plant lectins, artificial lectins and the like. Specific examples thereof include R-type lectin, calnexin / calreticulin, C-type lectin, galectin, legume lectin, L-type lectin, P-type lectin, annexin, and I-type lectin.
More specifically, for example, SBA (Soybean Agglutinin), LCA (Lens culinaris Agglutinin), AAL (Aleuria aurantia Lectin), UEA (Ulex europaeus Agglutinin), PNA (Peanut Agglutinin), WGA (Wheat Germ Agglutinin), Con A (Concanavalin A) and so on.

The antibody includes both a polyclonal antibody and a monoclonal antibody. The antibody may include antibodies of any mammalian origin and may further belong to any immunoglobulin class of IgG, IgA, IgM, IgD or IgE, but IgG is preferred. is there. As the antibody, a commercially available antibody that binds to the target surface molecule or an antibody stored in a research institution may be used. Alternatively, one of ordinary skill in the art can prepare an antibody according to a conventionally known method.
In addition to the above-mentioned polyclonal antibody, natural antibody such as monoclonal antibody (mAb), chimeric antibody that can be produced by using gene recombination technology, humanized antibody, and single-stranded antibody, these antibodies can be used as antibodies. Contains fragments of. The antibody fragment means a region of a part of the antibody described above, and specifically includes Fab, Fab', F (ab') 2, scAb, scFv, scFv-Fc and the like.

Examples of the cell-cell adhesion factor include integrins and the like. As the integrin, for example, a heterodimer composed of two subunits of α chain and β chain is preferably used. For example, integrin α1β1, α2β1, α3β1, α6β1, α7β1, α6β4, α10β1, α11β1, αLβ2, αMβ2, αXβ2, αDβ2, α5β1, αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, αIIbβ3, α4β1, α4β7, α9β1, αDβ2, αLβ2, αMβ2, αXβ2, αEβ7, etc.

As described above, the ligand B used in the present embodiment requires a metal ion C for binding to the analyze A.
At the time of production, such a ligand B is adjusted to be bound to the metal ion C, and can be bound to the metal ion A immediately after the addition of the analite A.
However, as shown in FIG. 2, when the Analyte A dissociates from the Ligand B, it is considered that not only the Analyte A but also the metal ion C dissociates from the Ligand B.
Therefore, in order to reuse the biochip 1 that has been used once for the next analysis, the allite A is removed, and the ligand B is supplied with the metal ion C necessary for binding to the next allite A. Therefore, it is necessary to return the biochip 1 to the same initial state as at the time of manufacture.
Therefore, in the distribution mechanism 3 according to the present embodiment, after the sample solution is circulated, the allite A is removed from the ligand B in the flow path 2, and the metal ion C is added to the ligand B. It is configured to circulate the solution to be supplied (hereinafter, also referred to as a regenerated solution).

The regenerated solution is, for example, a buffer solution or the like containing a salt of the metal ion C, distilled water, or the like, and it is preferable to use a solution having a salt concentration of 1 mol / L or more and 5 mol / L or less, and 3 mol. More preferably, it is / L or more and 4 mol / L or less.

As the metal ion C contained in the regenerated solution, those required by the ligand B may be appropriately used, and various metal ions C such as monovalent, divalent or trivalent metal ion C are used. be able to.

Since it is considered that the ionic radius may be related to the binding between the ligand B and the metal ion C, for example, when a lectin, an antibody or an intercellular adhesion factor is used as the ligand B. If there is, since a plurality of divalent metal ions have been reported as the metal ions C required for the binding of these ligands B to the analyze A, the metal ions C are divalent or higher. Can be inferred to be preferable.
The metal ion C preferably has an ionic radius of 1.5 Å or less, and more preferably 1.35 Å or less.
Further, the salt of the metal ion C is preferably highly soluble in water in order to be adjusted as an aqueous solution having the salt concentration as described above. For example, a salt that dissolves 30 g / 100 ml or more is preferable, and a salt that dissolves 50 g / 100 ml or more is more preferable.
Specific examples of such metal ions include magnesium ion, manganese ion, calcium ion, strontium ion, barium ion and the like. Examples of the salts of these metal ions include halides such as chlorides and sulfates.
These divalent metal ions have a relatively small ionic radius and can be incorporated into the protein structure and interact with hydrated water. When the hydrated water bound to the protein interacts with the metal ion, the position of the hydrated water moves, and it is considered that the structure of the protein changes.
Among these, magnesium ion and manganese ion have relatively small ionic radii of 0.65 Å and 0.72 Å, respectively. Therefore, for example, calcium ion (0.99 Å), strontium ion (1.13 Å) or barium ion (1). It is conceivable that it interacts with one or more more hydrated water than in the case of .35 Å).
As a result, it is considered that magnesium ions and manganese ions are more likely to affect the structural change of proteins than metal ions having a relatively large ionic radius such as calcium ions. Further, for example, magnesium chloride or manganese chloride is known to have very high solubility in water, 54.3 g / 100 ml and 72.3 g / 100 ml, respectively.
For these reasons, magnesium ion or manganese ion is considered to be particularly preferable as the metal ion C.

The procedure and process of the interaction detection method using the interaction detection device 100 according to the present embodiment will be described below with reference to FIG.
First, the biochip 1 is arranged in the flow path 2, and distilled water or phosphate buffered saline (PBS), which is a mobile phase, is circulated in the flow path 2 at a constant flow rate to flow the flow path 2. The conditions inside are stabilized (condition stabilization step, S1 in FIG. 3).

Next, a certain amount of data is injected into the flow path 2 from the sample introduction port.
After that, the mobile phase is allowed to flow at a constant flow rate for a predetermined time, and the interaction between the ligand B and the allite A during the flow of the mobile phase is detected by the detection unit 4 (detection step, S2 in FIG. 3). ).

After the detection by the detection unit 4 is completed, the regenerated solution is circulated through the flow path 2 by the distribution mechanism 3 at a constant flow rate for a predetermined time (regeneration step, S3 in FIG. 3).
After this regeneration step, the condition stabilization step described above may be performed, or the condition stabilization step may be omitted, the next sample may be injected from the sample inlet, and the next analysis may be started. ..

According to the interaction detection device 100 and the interaction detection method configured in this way, the following effects can be obtained.
After using the biochip 1 for analysis, the biochip 1 can be returned to the initial state simply by supplying the regenerated solution to the biochip 1, so that the time until the next analysis is started can be as long as possible. Can be shortened. According to the interaction detection method according to the present embodiment, a particularly remarkable effect can be exhibited when a plurality of samples are continuously measured.

Since the concentration of the salt containing the metal ion C in the regenerated solution is 1 mol / L or more and 5 mol / L or less, the ligand B is not irreversibly modified and is analyzed under mild conditions. A can be removed.

According to the interaction analysis method according to the present embodiment, the entire exosome can be analyzed without destroying the exosome, so that the exosome surface molecule, which is an indicator of the disease appearing on the cell surface, is not destroyed as much as possible. It can be analyzed as it is.

By providing the display unit, the apparatus of the present invention can confirm the color tone change of the portion where the ligand B is not immobilized on the surface of the biochip 1, and thus confirms the presence or absence of non-specific binding of the exosome. be able to.

The present invention is not limited to the above embodiment.
For example, in the above-described embodiment, an example in which a flow-type SPRi device is used has been described, but the present invention is not limited to this, and detection using the surface plasmon resonance phenomenon may be performed by a batch method.
Furthermore, it is not limited to those utilizing the surface plasmon resonance phenomenon, and immunologically detecting the binding between the analyst and the ligand by chemical or physical means using an antigen-antibody reaction, a labeling substance, or the like, for example, ELISA. Etc. may be used.

The ligand is not limited to the above-mentioned ligand, and may be any ligand as long as it contains a metal ion C for binding to the analyte.
It is not necessary that only one type of ligand is immobilized on one biochip, and a plurality of types of ligands may be immobilized. At least one of these plurality of ligands may be one that requires a metal ion C for binding to an analyte.
By immobilizing multiple pairs of ligands at different positions on one biochip, for example, sugar chains expressed on the surface of exosomes and surface antigens can be simultaneously detected in a single analysis. For example, cancer diagnosis and the like can be performed more quickly.
For example, a biochip regeneration kit containing the above-mentioned ligand-immobilized biochip and a regeneration solution may be used. In addition to the above-mentioned ones, this regeneration kit may contain various reagents such as PBS and PBS + 0.1% Casein.
As long as it does not contradict the gist of the present invention, various modifications and combinations of embodiments may be performed.

In this example, the surface molecules of human serum-derived exosomes obtained by diluting an exosome fraction obtained by ultracentrifugation from human serum (Human Serum Bio west Inc., S4200-100) 1000-fold or 100-fold were used as an antibody. The interaction with the ligand antibody or lectin was detected, the used biochip was regenerated with a regeneration solution containing metal ions, and then the interaction between the surface molecules of human serum-derived exosomes and each ligand was detected again. ..

<Devices and biochips used>
Interaction detection is performed by microarray type SPRi device (HORIBA, Ltd .: XelPleX) and biochip dedicated to the device (HORIBA, Ltd .: CS-HD; bio-immobilized carboxy group activated by succinimide. This was done using a chip).
Antibodies and lectins were used as ligands to be immobilized on this biochip.
As the antibody, a monoclonal antibody Anti-CD81 (Santa Cruz Biotechnology Inc., sc-166029) was used.
As the lectin, Maackia amurensis (MAM; J-Chemical, Inc., J117) was used.
The antibody was adjusted to a concentration of 0.1 mg / ml, and the lectin was dissolved in a 0.1% aqueous gelatin solution to a concentration of 2 mg / ml. These were spotted on the surface of the biochip by 10 nL each using a spotter. Then, these ligands were solid-phased on the surface of the base material by allowing to stand for 16 hours.

<Analysis procedure>
The surface of the chip was conditioned by sending PBS + 0.1% Casein as a mobile phase at a flow rate of 25 μL / min to the flow path containing the biochip described above.
With the reflectance at the time of stabilization set to 0%, the exosome suspended in PBS + 0.1% Casein was injected with an auto-injector, and then the solution was sent at a flow rate of 25 μL / min for 480 seconds, and then only PBS + 0.1% Casein was immediately administered. The liquid was sent at a flow rate of 25 μL / min for 480 seconds, and the reflectance was measured over time.
Then, a 4 mol / L MgCl ₂ aqueous solution as a regeneration solution was sent to the flow path at a flow rate of 200 μL / min for 100 seconds to regenerate the biochip.

<Results and discussion>
The change in reflectance by the above-mentioned procedure is shown in FIG. In the figure, the solid line graph shows the case where canine lectin (MAM) is used as a ligand, and the broken line graph shows the case where Anti-CD81 is used as a ligand.
Moreover, the SRP image in the same experiment is shown in FIG.
From the results of FIG. 5, it can be seen that in the detection step (steps (I) and (III) of FIG. 4), the ligand on the biochip and the surface molecule of the exosome, which is an analyst, interact with each other. .. Further, in the subsequent regeneration steps (steps (II) and (IV) of FIG. 4), the analysts were no longer detected, so it was found that the analysts were removed by the regeneration solution. Note that (I) to (IV) in FIG. 5 represent the respective steps (I) to (IV) in FIG.
Furthermore, when the exosomes were re-supplied to the biochip regenerated in this way, it was confirmed that even a ligand requiring a metal ion could detect the interaction as in the initial analysis.
In the same experimental operation, when, for example, PBS described in Prior Document 1 is used as the regenerating solution, the analyte is removed from the ligand, but the metal ions required for binding are not supplied. Interactions may not be detected during the next analysis.
Further, in this example, magnesium chloride containing magnesium ion was used as the metal ion, but it is considered that the same result can be obtained even if this metal ion is used as another divalent ion such as manganese ion or calcium ion. Be done. Especially for manganese on, it can be expected that almost the same result will be obtained because the ionic radius is close to that of magnesium ion.

100 ... Interaction detection device 1 ... Biochip 2 ... Flow path 3 ... Distribution mechanism 4 ... Detection unit 4
A ... Analite B ... Ligand C ... Metal ion

Claims (8)

A detection step in which a sample containing an exosome, which is an analite, is brought into contact with a ligand to detect an interaction, and
An interaction detection method including a regeneration step of returning the ligand to a state before contact with the sample.
The ligand requires a metal ion to bind to the analyze,
An interaction detection method, which comprises removing the allite from the ligand and using a solution for supplying the metal ion to the ligand in the regeneration step. The interaction detection method according to claim 1, wherein the metal ion contains a divalent metal ion. The interaction detection method according to claim 1 or 2, wherein the metal ion contains magnesium ion or manganese ion. The interaction detection method according to any one of claims 1 to 3, wherein the ligand contains at least one of a lectin, an antibody, and a cell adhesion factor. The interaction detection method according to any one of claims 1 to 4, wherein a surface plasmon resonance phenomenon is used. The interaction according to any one of claims 1 to 5, wherein the solution contains a salt of the metal ion, and the concentration of the salt is 1 mol / L or more and 5 mol / L or less. Action detection method. A biochip with a ligand immobilized on the surface of the substrate,
A flow path for accommodating the biochip inside and
A distribution mechanism for distributing a sample solution containing an exosome, which is an analite, in the flow path,
An interaction detection device including a detection unit for detecting an interaction between the ligand and the analyst on the surface of the biochip.
The ligand requires a metal ion to bind to the analite.
The distribution mechanism is characterized in that after the sample solution is circulated, a solution for removing the allite from the ligand and supplying the metal ion to the ligand is circulated in the flow path. Interaction detector. A biochip in which a ligand that requires a metal ion for binding to an exosome, which is an analite, is immobilized on the surface of a substrate, and a biochip.
A biochip regeneration kit containing a solution for removing the allite from the ligand and supplying the metal ion to the ligand.

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