JP2023134405A - Diluent for microchannel devices - Google Patents

Abstract

To provide a diluent which allows a test target biological substance to pass through a through-hole formed in a detection device.SOLUTION: A diluent for diluting a sample used for detecting a test target biological substance is provided, the diluent containing a nonionic surfactant that improves permeability of the test target biological substance through a through-hole in a detection device, where the nonionic surfactant may be at least either of an acetylene glycol surfactant and a polyalkylene glycol surfactant.SELECTED DRAWING: Figure 1A

Description

The present invention relates to a diluent suitably used to dilute a specimen used for detection of a test biological substance, and a coating composition suitably used to improve detection sensitivity of a test biological substance. Furthermore, the present invention relates to a test liquid, a method for preparing the test liquid, and the like.

As methods for identifying test biological substances such as viruses and bacteria, the PCR method for identifying genes, the method using a DNA chip, the ELISA method using an antigen-antibody reaction, and the like are well known. Attempts have also been made to detect viruses and bacteria by determining the size of particles in a sample.

In recent years, as a new identification method, a method has been proposed in which the time change in ionic current when particles to be measured in an electrolytic solution pass through a through hole is measured (for example, Patent Documents 1 to 3).

Special Publication No. 2014-521962 International Publication No. 2013/137209 pamphlet International Publication No. 2020/230219 pamphlet

However, these methods have the problem that measurement cannot be performed or the sensitivity of measurement is reduced because the biological substance to be measured, which is the particle to be measured, cannot pass through the through-hole formed in the detection device. Factors that may prevent passage through the through-hole include the test biological substance blocking the through-hole, the test liquid containing the test biological substance not spreading into the through-hole, and air bubbles in the test liquid blocking the through-hole. Examples include blocking.

The present invention has been made to solve the above problems, and provides a diluent and a test liquid that allow a biological substance to be tested to pass through a through hole formed in a detection device. The present invention aims to provide a coating composition for improving the detection sensitivity of biological substances to be tested. A further object of the present invention is to provide a method for preparing a test solution using the diluent.

In order to solve the above-mentioned problems, the present inventors conducted intensive studies and found that the above-mentioned problems could be solved, and completed the present invention having the following gist.
That is, the present invention includes the following.
[1] A diluent for diluting a specimen used for detection of a biological substance to be tested, and a nonionic surfactant that improves permeability of the biological substance to be tested into a through-hole in a detection device. , wherein the nonionic surfactant is at least one of an acetylene glycol surfactant and a polyalkylene glycol surfactant.
[2] A nonionic surfactant that is a diluent for diluting a specimen used for detection of a biological substance to be tested and improves the permeability of the biological substance to be tested into a through-hole in a detection device. A diluent containing the following, wherein the nonionic surfactant is represented by the following formula (1).
A ¹ -B-A ² ...Formula (1)
(In formula (1), A ¹ and A ² each independently represent a monovalent group exhibiting hydrophilicity, and B represents a divalent group exhibiting relative hydrophobicity compared to A ¹ and A ² . (Represents an organic group.)
[3] The diluent according to [1] or [2], wherein the capillary force given by the following formula (F1) or (F2) in the microchannel is negative.
(γ in formulas (F1) and (F2) represents the surface tension of the diluted liquid, and θ represents the contact angle of the diluted liquid. Also, d in formula (F1) is the short side of the rectangular flow path, ω represents the long side of the rectangular flow path, and r in equation (F2) represents the radius of the circular flow path.)
[4] The diluent according to [3], wherein the inner surface of the microchannel is made of resin, ceramics, semimetal, or metal.
[5] The diluent according to any one of [1] to [4], further containing an aqueous solution.
[6] The diluent according to [5], wherein the aqueous solution is selected from physiological saline and phosphate buffered saline.
[7] The diluent according to any one of [1] to [6], wherein the biological substance to be tested is at least one of bacteria, viruses, nucleic acids, extracellular vesicles, and proteins.
[8] Any one of [1] to [7], wherein the detection of the test biological material is performed by the test biological material passing through the through hole formed in the detection device. Diluent as indicated.
[9] A test liquid containing a specimen used for detecting a biological substance to be tested and a nonionic surfactant that improves permeability of the biological substance to be tested into a through-hole in a detection device, , a test liquid in which the nonionic surfactant is at least one of an acetylene glycol surfactant and a polyalkylene glycol surfactant.
[10] A test liquid containing a specimen used for detecting a biological substance to be tested and a nonionic surfactant that improves permeability of the biological substance to be tested into a through-hole in a detection device, , a test liquid in which the nonionic surfactant is represented by the following formula (1).
A ¹ -B-A ² ...Formula (1)
(In formula (1), A ¹ and A ² each independently represent a monovalent group exhibiting hydrophilicity, and B represents a divalent group exhibiting relative hydrophobicity compared to A ¹ and A ² . (Represents an organic group.)
[11] The test liquid according to [9] or [10], wherein the capillary force given by the following formula (F1) or (F2) in the microchannel is negative.
(γ in formulas (F1) and (F2) represents the surface tension of the test liquid, and θ represents the contact angle of the test liquid. Also, d in formula (F1) is the short side of the rectangular flow path, ω represents the long side of the rectangular flow path, and r in equation (F2) represents the radius of the circular flow path.)
[12] Any one of [9] to [11], wherein the detection of the test biological material is performed by the test biological material passing through the through hole formed in the detection device. Test solution as described.
[13] A method for preparing a test liquid, comprising mixing a specimen used for detection of a biological substance to be tested and the diluent according to any one of [1] to [7].
[14] A coating composition for improving the detection sensitivity of a test biological substance, the composition comprising a nonionic surfactant that improves the permeability of the test biological substance to a through-hole in a detection device. a coating composition, wherein the nonionic surfactant is at least one of an acetylene glycol surfactant and a polyalkylene glycol surfactant.
[15] A coating composition for improving the detection sensitivity of a biological substance to be tested, the composition comprising a nonionic surfactant that improves the permeability of the biological substance to be tested into a through-hole in a detection device. A coating composition, wherein the nonionic surfactant is represented by the following formula (1).
A ¹ -B-A ² ...Formula (1)
(In formula (1), A ¹ and A ² each independently represent a monovalent group exhibiting hydrophilicity, and B represents a divalent group exhibiting relative hydrophobicity compared to A ¹ and A ² . (Represents an organic group.)
[16] A diluent containing a nonionic surfactant for improving the permeability of biological substances passing through a flow path.
[17] The diluent according to [16], wherein the nonionic surfactant is represented by the following formula (1).
A ¹ -B-A ² ...Formula (1)
(In formula (1), A ¹ and A ² each independently represent a monovalent group exhibiting hydrophilicity, and B represents a divalent group exhibiting relative hydrophobicity compared to A ¹ and A ² . (Represents an organic group.)
[18] A coating composition that suppresses the adhesion of biological substances within a flow path, the coating composition containing a nonionic surfactant.
[19] The coating composition according to [18], wherein the nonionic surfactant is represented by the following formula (1).
A ¹ -B-A ² ...Formula (1)
(In formula (1), A ¹ and A ² each independently represent a monovalent group exhibiting hydrophilicity, and B represents a divalent group exhibiting relative hydrophobicity compared to A ¹ and A ² . (Represents an organic group.)

According to the present invention, there is provided a diluent and a test liquid that allow a test biological substance to pass through a through-hole formed in a detection device, and a test solution that can improve the detection sensitivity of a test biological substance. A coating composition can be provided. Further, according to the present invention, it is possible to provide a method for preparing a test solution using the diluent. Further, it is possible to provide a diluent for improving the permeability of biological substances passing through the channel, and a coating composition that suppresses the adhesion of biological substances inside the channel.

FIG. 2 is a schematic cross-sectional view of an example of a detection device. FIG. 1B is a schematic cross-sectional view of an example of a detection apparatus having the detection device of FIG. 1A. It is a cross-sectional schematic diagram of another example of a detection device.

Each term described in the present invention shall be construed as preferred in each embodiment.
(Diluted solution)
The diluent of the present invention contains a nonionic surfactant.

One embodiment of the diluent of the present invention is a diluent for diluting a specimen used for detection of a biological substance to be tested. The nonionic surfactant improves the permeability of the biological material to be tested into the through-holes in the detection device. "Permeability" refers to the ease with which a test biological material passes through a through-hole.
Detection of the biological material to be tested is performed, for example, by the biological material to be tested passing through a through hole formed in the detection device. Embodiments of the detection device will be described later.

When the diluent contains a nonionic surfactant, the test liquid obtained by diluting the specimen with the diluent becomes easier to be provided to the through-hole formed in the detection device. For example, the test liquid easily wets and spreads in and around the through hole formed in the detection device.
Furthermore, since the test liquid contains a nonionic surfactant, the test liquid is less likely to have bubbles.
As a result of one or more of these being involved, it becomes easier for the biological substance to be tested to pass through the through-hole. Further, the diluent of the present invention may be one that dilutes the biological material itself.

Further, an embodiment of the diluent of the present invention may be a diluent for improving the permeability of biological substances passing through the flow path. The flow path may be, for example, the flow path described in International Publication No. 2017/065279 pamphlet. In recent years, various devices have been developed that use microfabrication technology such as MEMS (Microelectromechanical Systems) technology to create channels and holes with predetermined shapes on the order of μm on a substrate (chip), and these devices are becoming increasingly popular in biotechnology and chemical engineering. It is used in experiments with small amounts, isolation, purification, analysis, etc.
For example, platelets can be produced using a bioreactor with a structure similar to the human spinal cord. Such a bioreactor includes a porous thin film in which a plurality of micropores are formed in a controlled shape and arrangement and megakaryocytes are arranged on the surface of one side thereof, and a porous thin film provided on the one side of the porous thin film, and A platelet comprising a culture chamber in which megakaryocytes are cultured, and a microchannel provided on the other side of the porous thin film, through which a culture solution flows while the megakaryocytes are cultured in the culture chamber. A production channel device has been reported. Such a reactor can produce platelets by applying shear stress to megakaryocytes through micropores.
However, devices using biological samples have problems in that cells and proteins derived from the biological sample adhere to the surface of the device, resulting in clogging and a decrease in analysis accuracy and sensitivity. In order to solve this problem, a microchannel device coated with a cell adhesion inhibitor containing a polymer having a repeating unit having a sulfinyl group in its side chain as an active ingredient has been reported. The diluent of the present invention has the effect of improving the permeability of biological substances passing through the above-mentioned flow path.

<Nonionic surfactant>
The nonionic surfactant is not particularly limited as long as it lowers the surface free energy of the test solution obtained by diluting the specimen with a diluent.
Nonionic surfactants are less likely to alter biological materials. For example, nonionic surfactants are less likely to disrupt the viral envelope.

Furthermore, as a nonionic surfactant, a block containing a hydrophilic group or a block containing a monovalent group having a hydrophilic group is referred to as the A block, and a divalent surfactant that exhibits relative hydrophobicity compared to the A block. If a block having an organic group is a B block, a nonionic surfactant expressed in the format ABA, that is, an organic group exhibiting hydrophobicity (B block) is an A block exhibiting hydrophilicity. Sandwiched type compounds are preferred. The two A blocks may have the same structure or different structures.

（式（Ａ）中、Ｒ^１１は、それぞれ独立して、親水性基で置換されていてもよい炭素原子数２～６のアルキレン基を表す。Ｘは、ヒドロキシ基、アミノ基又は炭素数原子数１～６のアルコキシ基を表す。ｎは、０以上の整数を表す。＊は結合手を表す。）
ｎは、例えば、０～３０の整数を表す。
炭素原子数１～６のアルコキシ基としては、メトキシ基が好ましい。 <A block>
The monovalent group for the A block is preferably a monovalent organic group having a repeating unit having an alkylene glycol unit structure. At least one of the hydrogen atoms contained in the alkylene glycol may be substituted with a hydrophilic group, and preferably the hydrophilic group is present at the end of the A block that is farthest from the bonding portion with the B block.
Specific examples of the hydrophilic group of the substituent and the hydrophilic group of the terminal portion include, for example, a hydroxy group, an amino group, and an alkoxy group having 1 to 6 carbon atoms. As the alkoxy group having 1 to 6 carbon atoms, a methoxy group is preferred.
Examples of the A block include a monovalent group having a structure represented by the following formula (A).

(In formula (A), R ¹¹ each independently represents an alkylene group having 2 to 6 carbon atoms which may be substituted with a hydrophilic group. X is a hydroxy group, an amino group, or an atom having a carbon number Represents an alkoxy group of numbers 1 to 6. n represents an integer greater than or equal to 0. * represents a bond.)
n represents an integer from 0 to 30, for example.
As the alkoxy group having 1 to 6 carbon atoms, a methoxy group is preferred.

（式（Ｂ）中、Ｒ^１２は、それぞれ独立して、炭素原子数３～１０のアルキレン基を表す。ｎは、２以上の整数を表す。＊は結合手を表す。）
ｎは、例えば、２～３０の整数を表す。 <B block>
The organic group for the B block is not limited as long as it is a divalent organic group that is relatively hydrophobic compared to the A block, but preferably a repeating unit having an alkylene glycol unit structure. An organic group which is a divalent organic group having the following and which is not substituted with the above-mentioned hydrophilic group is preferable.
Examples of the B block include a divalent organic group having a structure represented by the following formula (B).

(In formula (B), R ¹² each independently represents an alkylene group having 3 to 10 carbon atoms. n represents an integer of 2 or more. * represents a bond.)
n represents an integer from 2 to 30, for example.

Examples of the nonionic surfactant include a nonionic surfactant represented by the following formula (1).
A ¹ -B-A ² ...Formula (1)
(In formula (1), A ¹ and A ² each independently represent a monovalent group exhibiting hydrophilicity, and B represents a divalent group exhibiting relative hydrophobicity compared to A ¹ and A ² . (Represents an organic group.)

Examples of A ¹ and A ² in formula (1) include a monovalent group represented by the above formula (A), a hydroxyl group, and the like.

（式（Ｃ）中、Ｒ^２１、Ｒ^２２、Ｒ^２３、及びＲ^２４は、それぞれ独立して、水素原子、又は炭素原子数１～３０のアルキル基を表す。）
なお、式（Ｃ）で表されるグリコールは、２つのヒドロキシ基がＡ^１及びＡ^２に相当する式（１）で表される非イオン性界面活性剤でもある。 B in formula (1) is, for example, a divalent organic group represented by the above formula (B), a divalent organic group obtained by removing two hydroxy groups from alkenylene glycol, or a divalent organic group obtained by removing two hydroxy groups from alkynylene glycol. Examples include divalent organic groups excluding groups.
Alkenylene glycol is a compound in which one of the carbon-carbon single bonds of alkylene glycol is replaced with a carbon-carbon double bond.
Alkynylene glycol is a compound in which one carbon-carbon single bond of alkylene glycol is replaced with a carbon-carbon triple bond, and is also called acetylene glycol.
Examples of acetylene glycols include glycols represented by the following formula (C).

(In formula (C), R ²¹ , R ²² , R ²³ , and R ²⁴ each independently represent a hydrogen atom or an alkyl group having 1 to 30 carbon atoms.)
In addition, the glycol represented by formula (C) is also a nonionic surfactant represented by formula (1), in which two hydroxy groups correspond to A ¹ and A ² .

Examples of nonionic surfactants include polyoxyalkylene alkyl ether (polyoxyethylene alkyl ether, polyoxypropylene alkyl ether, polyoxyethylene polyoxypropylene alkyl ether), polyoxyethylene glycerin fatty acid ester, polyoxyethylene sorbitan Fatty acid ester, polyoxyethylene sorbitol fatty acid ester, polyglycerin fatty acid ester, glycerin fatty acid ester, sorbitan fatty acid ester, polyethylene glycol fatty acid ester, propylene glycol monofatty acid ester, alkyl polyhydric alcohol ether, polyoxyalkylene block polymer, polyoxyethylene alkyl Phenyl ether, polyoxyethylene alkylamine, fatty acid alkanolamide, acylmethyl glucamide, polyoxyethylene acetylene glycol, polyoxyethylene lanolin, polyoxyethylene lanolin alcohol, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxy Examples include ethylene phytosterol, polyoxyethylene cholestanol, polyoxyethylene nonylphenyl formaldehyde condensate, and alkyl glucoside.
Among these, polyoxyethylene acetylene glycol (acetylene glycol surfactant) and polyoxyalkylene block polymer (polyalkylene glycol surfactant) are preferred.

（式（２）中、Ｒ^１、Ｒ^２、Ｒ^３、及びＲ^４は、それぞれ独立して、水素原子、又は炭素原子数１～１０のアルキル基を表す。Ｒ^１とＲ^２は、直接若しくはヘテロ原子を介して、一緒になって、３員環から８員環の環状構造を形成していてもよい。また、この環状構造は、官能基を有していてもよい。Ｒ^３とＲ^４は、直接若しくはヘテロ原子を介して、一緒になって、３員環から８員環の環状構造を形成していてもよい。また、この環状構造は、官能基を有していてもよい。Ｒ^５及びＲ^６は、それぞれ独立して、水素原子、又は炭素原子数１～６のアルキル基を表す。
ｍ及びｎは、それぞれ、０以上の整数である）。
ヘテロ原子としては、例えば、酸素原子、窒素原子、硫黄原子などが挙げられる。
官能基としては、例えば、ヒドロキシ基、アミノ基などが挙げられる。
ｍ及びｎは、例えば、それぞれ０～２５の整数であり、ｍ＋ｎは０～４０である。
炭素原子数１～６のアルコキシ基としては、メトキシ基が好ましい。 The acetylene glycol surfactant is represented by the following formula (2), for example.

(In formula (2), R ¹ , R ² , R ³ , and R ⁴ each independently represent a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. R ¹ and R ² directly represent Alternatively, they may be joined together via a heteroatom to form a 3- to 8-membered cyclic structure.Also, this cyclic structure may have a functional group.R ³ and R ⁴ may be combined directly or via a hetero atom to form a 3- to 8-membered cyclic structure.Also, this cyclic structure may have a functional group. Good. R ⁵ and R ⁶ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
m and n are each an integer greater than or equal to 0).
Examples of the heteroatom include an oxygen atom, a nitrogen atom, and a sulfur atom.
Examples of the functional group include a hydroxy group and an amino group.
For example, m and n are each integers from 0 to 25, and m+n is from 0 to 40.
As the alkoxy group having 1 to 6 carbon atoms, a methoxy group is preferred.

The acetylene glycol surfactant may be a commercially available product. Examples of commercially available acetylene glycol surfactants include Surfynol (registered trademark) or Olfine (registered trademark) manufactured by Nissin Chemical Industries, Ltd., and Acetylenol (registered trademark) manufactured by Kawaken Fine Chemicals.

（式（２－１）～式（２－４）中、Ｒ^１は、水素原子、高級アルコール残基、アルキルフェノール残基、アリールフェノール残基、脂肪酸残基、高級脂肪族アミン残基又は脂肪酸アミド残基を表す。ｘ１、ｘ２、ｘ３及びｘ４は、それぞれ独立して４以上の整数を表す。ｙ１、ｙ２、ｙ３及びｙ４は、それぞれ独立して、１６以上の整数を表す。）
ｘ１、ｘ２、ｘ３及びｘ４は、例えば、それぞれ独立して、４～９００の整数を表す。
ｙ１、ｙ２、ｙ３及びｙ４は、例えば、それぞれ独立して、１６～１３０の整数を表す。 The polyalkylene glycol surfactant contains, for example, ethylene glycol and propylene glycol in block form. Such polyalkylene glycol surfactants are represented by the following formulas (2-1) to (2-4), for example.

(In formulas (2-1) to (2-4), R ¹ is a hydrogen atom, a higher alcohol residue, an alkylphenol residue, an arylphenol residue, a fatty acid residue, a higher aliphatic amine residue, or a fatty acid amide) Represents a residue. x1, x2, x3 and x4 each independently represent an integer of 4 or more. y1, y2, y3 and y4 each independently represent an integer of 16 or more.)
x1, x2, x3 and x4 each independently represent an integer from 4 to 900, for example.
For example, y1, y2, y3 and y4 each independently represent an integer from 16 to 130.

Higher alcohol residues are linear or branched alcohol residues having 4 to 18 carbon atoms, such as butyl alcohol residues, hexyl alcohol residues, ethylhexyl alcohol residues, lauryl alcohol residues, and oleyl alcohol residues. Examples include residues. Among these, ethylhexyl alcohol residues and lauryl alcohol residues are preferred.
Alkylphenol residues are phenol residues to which the same or different linear or branched alkyl groups having 1 to 3 carbon atoms are bonded, such as tributylphenol residues and ethylhexylphenol residues. , nonylphenol residues, etc. Among these, nonylphenol residues are preferred.
The arylphenol residue is a phenol to which 1 to 3 styryl groups are bonded, and to which the same or different 1 or 2 straight chain or branched alkyl group having 1 to 4 carbon atoms may be bonded. Examples of the residue include monostyrylphenol residue, distyrylphenol residue, tristyrylphenol residue, distyrylcresol residue, and the like. Among these, tristyrylphenol residues are preferred.
The fatty acid residue is a saturated or unsaturated fatty acid residue having 2 to 18 carbon atoms, and examples thereof include acetic acid residue, butyric acid residue, lauric acid residue, and oleic acid residue. Among these, lauric acid residues are preferred.
Higher aliphatic amine residues are saturated or unsaturated aliphatic amine residues having 4 to 18 carbon atoms, such as hexylamine residues, ethylhexylamine residues, laurylamine residues, oleylamine residues, etc. can be mentioned. Among these, lauric amine residues and oleyl amine residues are preferred.
The fatty acid amide residue is a saturated or unsaturated fatty acid amide residue having 4 to 18 carbon atoms, and includes, for example, a butyric acid amide residue, a lauric acid amide residue, an oleic acid amide residue, and the like. Among these, lauric acid amide residues are preferred.

In formula (2-1) and formula (2-2), those in which R ¹ is a hydrogen atom are nonionic surfactants called pluronic type, and also those in formula (2-3) and formula (2 The polyalkylene glycol surfactant represented by -4) is a nonionic surfactant called Tetronic type.

In formulas (2-1) and (2-2), R ¹ is preferably a hydrogen atom.

The number average molecular weight of the propylene glycol moiety (PO-added moiety) in the polyalkylene glycol surfactant is preferably 2,000 to 4,000, more preferably 2,500 to 3,500, and 2,700 to 3,200. Particularly preferred.
In the polyalkylene glycol surfactant, the ratio of the number average molecular weight of the ethylene glycol moiety (EO-added moiety) to the number-average molecular weight of the propylene glycol moiety (PO-added moiety) is preferably 0.05 to 10 times, and 0.05 to 10 times. It is more preferably 1 to 4 times, and particularly preferably 0.2 to 1 times.

The polyalkylene glycol surfactant may be a commercially available product. Commercially available polyalkylene glycol surfactants include, for example, Adeka Pluronic manufactured by ADEKA, Pluronic (registered trademark) manufactured by BASF, Unilube (registered trademark) manufactured by NOF Corporation, and the like.

These nonionic surfactants can be used alone or in combination of two or more.

The content of the nonionic surfactant in the diluted solution is not particularly limited, but is preferably 0.001% by mass to 5% by mass, more preferably 0.01% by mass to 3% by mass, and 0.03% by mass. ~1% by weight is particularly preferred.

<Aqueous solution>
The diluent preferably contains an aqueous solution as another component.
Examples of aqueous solutions include physiological saline, phosphate buffer, citrate buffer, citric acid/phosphate buffer, acetate buffer, borate buffer, tartrate buffer, Tris buffer, Tris-HCl buffer, Examples include phosphate buffered saline, citric acid/phosphate buffered saline, and the like.
Among these, physiological saline and phosphate buffered saline are preferred in terms of availability and pH and salt composition close to body fluids.

The diluted solution can be prepared, for example, by mixing an aqueous solution and a nonionic surfactant at a predetermined ratio.

（式（Ｆ１）及び（Ｆ２）中のγは希釈液又は試験液の表面張力を表し、θは希釈液又は試験液の接触角を表す。また、式（Ｆ１）中のｄは矩形流路の短辺、ωは矩形流路の長辺を表し、式（Ｆ２）中のｒは円形流路の半径を表す。） <Capillary force of diluent and test solution>
The capillary force of the diluent and test liquid is not particularly limited, but from the viewpoint of suitably obtaining the effects of the present invention, the capillary force given by the following formula (F1) or (F2) in the microchannel is negative. is preferred. Since the capillary force is negative, the diluent and the test liquid can easily flow through the microchannel.
Note that equation (F1) is an equation for calculating capillary force in a rectangular flow path. Equation (F2) is an equation for calculating capillary force in a circular flow path.
These formulas are described in Journal of Japan Society for Precision Engineering Vol. 82, No. 5, 2016. is introduced in.

(γ in formulas (F1) and (F2) represents the surface tension of the diluted liquid or test liquid, and θ represents the contact angle of the diluted liquid or test liquid. Also, d in formula (F1) represents the rectangular flow path. The short side of ω represents the long side of the rectangular flow path, and r in equation (F2) represents the radius of the circular flow path.)

Surface tension can be measured, for example, by the following method.

[Surface tension measurement]
Add the diluent or test solution to the petri dish. Using a surface tension meter (Kyowa Interface Science Co., Ltd., DY-700), the surface tension is measured after 1 minute by the Willhelmy method.

The contact angle can be measured, for example, by the method under Condition 1 or 2 below.

[Contact angle measurement/condition 1]
4 μl of diluted solution or test solution is applied to a PET substrate (Lumirror Film T-60, Toray Industries, Inc.) using a pipette. The contact angle of the droplet 5 seconds after attachment is measured using a contact angle meter (Kyowa Interface Science Co., Ltd., DM-701).

[Contact angle measurement/condition 2]
4 μl of the diluent or test solution is applied to the SiN substrate using a pipette. The contact angle of the droplet 1 second after attachment is measured using a contact angle meter (Kyowa Interface Science Co., Ltd., DM-701).

In formula (F1), for example, the size of the rectangular microchannel made of PET (polyethylene terephthalate) is set such that the long side ω = 0.001 m and the short side d = 0.00054 m, and the capillary tube of each diluted liquid or test liquid is Calculate the force.
In equation (F2), for example, the capillary force of each diluted liquid or test liquid is calculated by setting the radius of a circular microchannel (for example, a through hole) made of SiN to radius r=0.00000015 m.

The capillary force determined by formula (F1) is, for example, more preferably -4.5×10 ⁶ mN/m ² or more and less than 0 mN/m ² .
The capillary force determined by formula (F2) is, for example, more preferably −2.0×10 ⁹ mN/m ² or more and less than 0 mN/m ² .

For example, the inner surface of the microchannel is made of resin, ceramics, semimetal, metal, or glass.

The resin may be either a natural resin or its derivative, or a synthetic resin.
Examples of natural resins or derivatives thereof include cellulose, cellulose triacetate (CTA), nitrocellulose (NC), and cellulose on which dextran sulfate is immobilized.
Examples of synthetic resins include polyacrylonitrile (PAN), polyester polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), Polyurethane (PU), ethylene vinyl alcohol (EVAL), polyethylene (PE), polyester, polypropylene (PP), polyvinylidene fluoride (PVDF), polyether sulfone (PES), polycarbonate (PC), polyvinyl chloride (PVC), Examples include polytetrafluoroethylene (PTFE), ultra-high molecular weight polyethylene (UHPE), polydimethylsiloxane (PDMS), acrylonitrile-butadiene-styrene resin (ABS), Teflon (registered trademark), and various ion exchange resins.

Examples of ceramics include SiN, SiO ₂ , Al ₆ O ₁₃ Si ₂ , ZiO ₂ , SiC, and Al ₂ O ₃ .

Examples of semimetals include boron, silicon, germanium, arsenic, antimony, and tellurium.

Examples of metals include typical metals: (alkali metals: Li, Na, K, Rb, Cs; alkaline earth metals: Ca, Sr, Ba, Ra), magnesium group elements: Be, Mg, Zn, Cd, Hg. ; Aluminum group elements: Al, Ga, In; Rare earth elements: Y, La, Ce, Pr, Nd, Sm, Eu; Tin group elements: Ti, Zr, Sn, Hf, Pb, Th; Iron group elements: Fe, Co, Ni; earthen elements: V, Nb, Ta, chromium group elements: Cr, Mo, W, U; manganese group elements: Mn, Re; noble metals: Cu, Ag, Au; platinum group elements: Ru, Rh, Examples include Pd, Os, Ir, and Pt.

The inner surface of the microchannel may be made of one type of material or a combination of two or more types. Among these materials, glass, silicon, silicon oxide, SiN, polystyrene (PS), Teflon (registered trademark), cycloolefin polymer (COP), or polydimethylsiloxane (PDMS) alone, or two types selected from these A combination of the above is preferred, and glass, cycloolefin polymer (COP), or polydimethylsiloxane (PDMS) alone, or a combination of two or more selected from these is most preferred.

<Biological material and biological material to be tested>
Examples of biological substances and biological substances to be tested include bacteria, viruses, nucleic acids, extracellular vesicles, proteins, and cells.

The bacteria referred to in the present invention are not excluded if they are prokaryotes that do not have a cell nucleus. Specific examples include Escherichia coli, Salmonella, Campylobacter, and the like.
The virus referred to in the present invention may be either an enveloped virus (including vaccines and viral vectors) or a non-enveloped virus. The envelope is a membrane-like structure composed of lipids and proteins derived from host cells, as well as glycoproteins derived from viruses.

Enveloped viruses in the present invention include Flaviviridae, Togaviridae, Retroviridae, Coronaviridae, Filoviridae, Rhabdoviridae, Bunyaviridae, Orthomyxoviridae, Paramyxoviridae, and Arenaviruses. Yellow fever virus, dengue virus, Japanese encephalitis virus, St. Louis encephalitis, Murray Valley encephalitis, West Nile, Central European encephalitis, and Russian spring/summer encephalitis. , hepatitis C virus, rubella virus, Sindbis virus, chikungunya virus, eastern equine encephalitis virus, Seibu equine encephalitis virus, Venezuelan equine encephalitis virus, human T lymphotropic virus, human immunodeficiency virus, coronavirus, SARS coronavirus (SARS-CoV-1), MERS coronavirus, SARS-CoV-2, Marburg virus, Ebola virus, rabies virus, California encephalitis virus, Hunteran virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, influenza A virus Viruses, influenza B virus, influenza C virus, Togoto virus, Dori virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, Lassa virus, lymphocytic choriomeningitis virus, Junin virus, Machupo virus, Guanarito virus, hepatitis D virus, hepatitis B virus, human herpesvirus 1 (herpes simplex virus type 1), human herpesvirus 2 (herpes simplex virus type 2), human herpesvirus 3 (varicella/shingles virus), Examples include human herpesvirus 5 (cytomegalovirus), human herpesvirus 6, human herpesvirus 7, human herpesvirus 4 (EB virus), human herpesvirus 8, and variola virus. These vaccine uses and vector uses are also included in the rights of this application.

The enveloped virus referred to in the present invention is not limited to those that are infectious and pathogenic to humans. Enveloped viruses that are infectious or pathogenic to animals other than humans or plants are not excluded from the enveloped viruses referred to in the present application.

Viruses without an envelope as used in the present invention include Reoviridae, Caliciviridae, Picornaviridae, Astroviridae, Hepeviridae, Parvoviridae, Polyomaviridae, Papillomaviridae, and Adenoviridae. Specific examples include reovirus, rotavirus, Colorado tick fever virus, Norwalk virus, Sapporovirus, poliovirus, coxsackievirus group A, coxsackievirus group B, enterovirus, rhinovirus, hepatitis A virus, astrovirus, Examples include hepatitis E virus, B19 virus, JC virus, hyperpavilloma virus, feline calicivirus and human adenovirus. These vaccine uses and vector uses are also included in the rights of this application.

The non-enveloped viruses used in the present invention are not limited to those that are infectious and pathogenic to humans. Non-enveloped viruses that are infectious or pathogenic to animals other than humans or plants are not excluded from the non-enveloped viruses referred to in the present application.

Nucleic acids as used in the present invention include DNA and RNA.

Examples of the extracellular vesicles in the present invention include exosomes, microvesicles, apoptotic bodies, ectosomes, microparticles, secreted microvesicles, and the like.

Examples of proteins in the present invention include fibrinogen, bovine serum albumin (BSA), human albumin, various globulins, β-lipoproteins, various antibodies (IgG, IgA, IgM), peroxidase, various complements, various lectins, and fibronectin. , lysozyme, von Willebrand factor (vWF), serum γ-globulin, pepsin, ovalbumin, insulin, histones, ribonuclease, collagen, and cytochrome c.

Examples of sugars in the present invention include glucose, galactose, mannose, fructose, heparin, and hyaluronic acid.

Examples of cells in the present invention include fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, red blood cells, platelets, macrophages, monocytes, osteocytes, pericytes, dendritic cells, and keratinocytes. , adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatic parenchymal cells, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, stellate cells Glial cells, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells, mononuclear cells, embryonic stem cells (ES cells), embryonic tumor cells , embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, and Various cell lines (e.g., HCT116, Huh7, HEK293 (human embryonic kidney cells), HeLa (human cervical cancer cell line), HepG2 (human liver cancer cell line), UT7/TPO (human leukemia cell line), CHO (Chinese hamster) Ovarian cell line), MDCK, MDBK, BHK, C-33A, HT-29, AE-1, 3D9, Ns0/1, Jurkat, NIH3T3, PC12, S2, Sf9, Sf21, High Five, Vero), etc. .

Among these, bacteria, viruses, nucleic acids, extracellular vesicles, and proteins need to be detected using minute through-holes, and because they are difficult to pass through the through-holes, the effectiveness of the present invention is These are the obtained biological material and the test biological material.

For each of these substances, one type or a combination of two or more types is not excluded.

Detection of the biological material to be tested is performed, for example, by the biological material to be tested passing through a through hole formed in the detection device.
The diameter of the through hole in this specification may be, for example, 50 nm to 10 μm, 50 μm to 1 μm, 50 nm to 500 nm, or 100 nm to 500 nm. good. When the hole of the through-hole (the surface of the through-hole in the direction perpendicular to the direction in which the through-hole penetrates) is not a perfect circle, the diameter here means the diameter of the inscribed circle.

Furthermore, the biological substance to be tested may be bound to, for example, gold nanoparticles or polystyrene beads. Moreover, the above-mentioned biological substance to be tested may be the biological substance itself.

<Sample>
Specimens include, for example, saliva, digestive juices other than saliva (gastric juice, bile, pancreatic juice, intestinal juice), sweat, nasal mucus, amniotic fluid, milk, lymph, tissue fluid, body cavity fluid, blood, urine, skin, mucous membrane, stool, semen, Examples include tears, vaginal secretions, nasopharyngeal swabs, nasal swabs, soil, environmental water, environmental swabs, and atmospheric collection materials.
Environmental water refers to water collected from the environment, such as sewage, tap water, groundwater, domestic wastewater, industrial wastewater, public water bodies (rivers, lakes, ports, coastal waters, public ditches, irrigation canals, etc.) This includes water from water bodies (water bodies and waterways) used for public purposes.
An environmental swab is an instrument used for swabbing when testing for microorganisms in the environment. Examples of environmental swabs include sterile cotton swabs.
An air trap is an instrument that collects fine particles when measuring fine particles in the atmosphere. Examples of the air collection material include air collection bags, collection filters, and the like.

(Test liquid)
The test solution of the present invention contains a specimen used for detecting a biological substance to be tested and a nonionic surfactant.
The test liquid is, for example, an electrolyte.

When the test liquid contains a nonionic surfactant, the test liquid is easily provided to the through hole formed in the detection device. For example, the test liquid easily wets and spreads in and around the through hole formed in the detection device.
Furthermore, since the test liquid contains a nonionic surfactant, the test liquid is less likely to have bubbles.
As a result of one or more of these being involved, it becomes easier for the biological substance to be tested to pass through the through-hole.

Specific examples and preferred examples of the test biological material include, for example, the specific examples and preferred examples of the test biological material exemplified in the description of the diluent of the present invention.
Examples of the specimen include the specimens exemplified in the description of the diluent of the present invention.
Specific examples and preferred examples of the nonionic surfactant include, for example, the specific examples and preferred examples of the nonionic surfactant exemplified in the explanation of the diluent of the present invention.

The content of the nonionic surfactant in the test liquid is not particularly limited, but is preferably 0.001% by mass to 1.0% by mass, more preferably 0.01% by mass to 0.2% by mass, and Particularly preferred is .015% by weight to 0.1% by weight.

Detection of the biological material to be tested is performed, for example, by the biological material to be tested passing through a through hole formed in the detection device. Details of the detection device will be described later.

The method for preparing the test liquid is not particularly limited, but the following method for preparing the test liquid of the present invention is preferred.

(Preparation method of test solution)
The method for preparing a test solution of the present invention includes mixing a specimen used for detection of a biological substance to be tested and a diluent of the present invention.
The mixing method is not particularly limited, and for example, a known method of diluting the specimen can be used.

The mixing ratio of the sample and diluent is not particularly limited, but it is preferable that the volume ratio of the diluent to the sample is 1 to 10,000 times, and preferably 10 to 5,000 times. More preferred.

Specific examples and preferred examples of the test biological material include, for example, the specific examples and preferred examples of the test biological material exemplified in the description of the diluent of the present invention.
Examples of the specimen include the specimens exemplified in the description of the diluent of the present invention.

(Coating composition)
The coating composition of the present invention contains a nonionic surfactant.
One embodiment of the coating composition of the present invention is a coating composition for improving the detection sensitivity of a biological substance to be tested.
Detection of the biological material to be tested is performed, for example, by the biological material to be tested passing through a through hole formed in the detection device.

When the coating composition contains a nonionic surfactant, the test liquid passing through the surface coated with the coating composition is easily provided to the through-hole formed in the detection device. For example, the test liquid easily wets and spreads in and around the through hole formed in the detection device.
Furthermore, since the coating composition contains a nonionic surfactant, the test liquid that comes into contact with the surface coated with the coating composition is less likely to have bubbles.
As a result of one or more of these being involved, it becomes easier for the biological substance to be tested to pass through the through-hole.

Moreover, one embodiment of the coating composition of the present invention is a coating composition that suppresses the adhesion of biological substances within a flow path.

The nonionic surfactant contained in the coating composition is not particularly limited, and specific examples and preferred examples of the nonionic surfactant include, for example, the nonionic surfactant contained in the diluent of the present invention. Specific examples and preferred examples of surfactants are listed.

The content of the nonionic surfactant in the coating composition is not particularly limited, but is preferably 0.01% by mass to 5% by mass, more preferably 0.03% by mass to 3% by mass, and 0.05% by mass. Particularly preferred is % by weight to 1% by weight.

Components other than the nonionic surfactant contained in the coating composition are not particularly limited, but water is preferred.
The water content in the coating composition is not particularly limited, but is preferably 90% by mass to 99.99% by mass, more preferably 95% to 99.97% by mass, and more preferably 99% to 99% by mass. Particularly preferred is 95% by weight.

The total content of water and nonionic surfactant in the coating composition is not particularly limited, but is preferably 95% to 100% by mass, more preferably 99% to 100% by mass, and 99% by mass to 100% by mass. Particularly preferred is .5% to 100% by weight.

In addition to water, the coating composition may further contain an organic solvent (for example, alcohol and a water-soluble organic solvent (excluding alcohol)).

Examples of the alcohol include alcohols having 2 to 6 carbon atoms.
Examples of the alcohol include ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (t-amyl alcohol), 3-methyl-1 -butanol, 3-methyl-3-pentanol, cyclopentanol, 1-hexanol, 2-hexanol, 3-hexanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-1-butanol, 3, 3-dimethyl-2-butanol, 2-ethyl-1-butanol, 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pen Tanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol and cyclohexanol Can be mentioned. These can be used alone or in combination of two or more.

A water-soluble organic solvent refers to an organic solvent that can be mixed with water and alcohol in any proportion, and no separation occurs after mixing.
Examples of water-soluble organic solvents include ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, methyl cellosolve acetate, ethyl cellosolve acetate, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, propylene glycol, propylene glycol monomethyl ether, and propylene glycol monoethyl ether. , propylene glycol monomethyl ether acetate, propylene glycol propyl ether acetate, acetone, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, 1,3-dimethyl- 2-imidazolidinone is mentioned. These can be used alone or in combination of two or more.

The content ratio (weight ratio) of water and organic solvent is, for example, (water): (organic solvent) = 20:80 to 80:20, 30:70 to 70:30, and 40:60 to 60: 40 and 50:50.

(An example of a method for detecting a biological substance to be tested)
An example of the method for detecting a biological substance to be tested of the present invention includes at least a passing step, and may further include other steps as necessary.

<Passing process>
The passing step is a step of causing the biological substance to be tested to pass through a through hole formed within the detection device. Details of the detection device will be described later.
The passing step includes providing the through-hole with a test liquid of the present invention.

In a method for detecting a biological substance to be tested, for example, electricity is applied to a through hole or a partition wall around the through hole, and an electric signal is detected when the biological substance to be tested passes through the through hole, thereby detecting the biological substance to be tested. Perform detection.

(detection device)
The detection device is not particularly limited as long as it has a through hole for detecting the biological substance to be tested.
The detection device includes, for example, a substrate in which a through hole is formed, and a first chamber and a second chamber partitioned by the substrate.
The first chamber and the second chamber are communicated through a through hole.
An example of the passing step in an example of the method for detecting a biological substance to be tested of the present invention is, for example, providing a test liquid to a first chamber, and moving the test liquid from the first chamber to the second chamber by passing through a through hole. This is done by
In another example of the passing step in the method for detecting a biological substance to be tested of the present invention, for example, the test liquid is provided to the first chamber and the second chamber, and a through hole is formed from the first chamber to the second chamber. This is done by moving the biological material to be tested through the passage.

The material of the walls forming the first chamber and the second chamber is not particularly limited, and examples thereof include resin, glass, SiN, _Al2O3 , _and the like. Examples of the resin include polyethylene terephthalate.

Examples of the detection device include the single particle analysis device described in International Publication No. 2013/137209 pamphlet.
The single particle analysis device is the following analysis device.
A measurement container, a first chamber and a second chamber in which the measurement container is partitioned by an insulating partition wall, and a through hole that communicates with the first chamber and the second chamber opened in the partition wall. and a first electrode arranged in the first chamber, and a second electrode arranged in the second chamber, and between the first electrode and the second electrode. The object to be measured is supplied with electricity through the through hole of the partition wall, and a detection signal is measured when the particulate object to be measured in the first chamber passes from the through hole into the second chamber. A single particle analysis device that measures the shape of an object,
(a) When the diameter of the particle to be measured is a, the diameter of the through hole is d, and the thickness of the partition wall near the through hole is t, the following equation is obtained;
t<a<d≦100a
or (b) where the major axis of the particle to be measured is L, the minor axis is s, the diameter of the through hole is d, and the thickness of the partition wall near the through hole is t, the following equation is satisfied. ;
s<L,
s<d≦100s and t<L
A single particle analysis device that satisfies the following.

Moreover, as a detection device, the biological substance detection device described in International Publication No. 2017/183716 pamphlet can be mentioned, for example.
The biological substance detection device is the following biological substance detection device.
substrate,
a through hole formed in the substrate through which the test biological substance passes;
Molecules that are formed in the through-hole and interact with the test biological material passing through;
a first chamber member forming a first chamber filled with an electrolytic solution with at least one side of the substrate including the through hole;
a second chamber member forming a second chamber filled with an electrolyte with at least the surface including the through hole on the other surface side of the substrate;
A device for detecting a biological substance, which identifies a biological substance to be tested based on the waveform of an ionic current when the biological substance passes through a through hole.

Further, as a detection device, for example, a particle identification sensor described in International Publication No. 2020/230219 pamphlet can be mentioned.
The particle identification sensor is the following particle identification sensor.
a first channel formed in the first layer through which an electrolytic solution containing particles to be identified can flow;
a second flow path formed in a second layer through which the electrolytic solution can flow;
a through hole connecting the first flow path and the second flow path;
a first electrode disposed within the first flow path;
a second electrode disposed in the second flow path, and identifies the to-be-identified particle by measuring a time change in ionic current generated when the to-be-identified particle passes through the through-hole. identification sensor.

An example of a detection device will be explained using figures.
FIG. 1A is a diagram showing an example of a detection device 2. As shown in FIG. The detection device 2 shown in FIG. 1A includes a first chamber member 51 that can form a first chamber 5 filled with a test liquid with at least one side of the substrate 3 including the through hole 4; It includes at least a second chamber member 61 that can form a second chamber 6 filled with a test liquid with at least the surface including the through hole 4 on the surface side.
The first chamber member 51 and the second chamber member 61 are preferably formed of an electrically and chemically inert material, such as glass, sapphire, ceramic, resin, rubber, elastomer, SiO ₂ , SiN, Al, etc. ₂ O ₃ and the like.
The first chamber 5 and the second chamber 6 are formed to sandwich the through hole 4, and are formed so that the sample introduced into the first chamber 5 can pass through the through hole 4 and move to the second chamber 6. There are no particular restrictions. For example, the first chamber member 51 and the second chamber member 61 may be created separately and bonded to the substrate 3 so as to be liquid-tight. Alternatively, a substantially rectangular parallelepiped box member with one surface open may be formed, the substrate 3 may be inserted and fixed in the center of the box, and then the open surface may be liquid-tightly sealed. In that case, the first chamber member 51 and the second chamber member 61 do not mean separate members, but mean a part of a box member separated by the substrate 3. Although not shown, holes may be formed in the first chamber member 51 and the second chamber member 61 as necessary for filling and discharging the test liquid and for inserting electrodes and/or leads. good.
FIG. 1B is a schematic diagram showing an example of the test biological substance detection device 1. As shown in FIG. In addition to the detection device 2 shown in FIG. 1A, the test biological substance detection apparatus 1 includes a first electrode 52 formed at a location in contact with the test liquid in the first chamber 5, and a first electrode 52 in contact with the test liquid in the second chamber 6. It includes at least a second electrode 62 formed at a location and an ammeter 7 for measuring the ionic current when the biological substance 41 to be examined passes through the through hole 4 .
The test biological substance detection device 1 also includes an analysis section 8 for analyzing the ionic current measured by the ammeter 7, and for displaying the measured ionic current value and/or the results analyzed by the analysis section 8, as necessary. The display unit 9 may include a display unit 9, a program memory 10 in which a program for operating the analysis unit 8 and the display unit 9 is stored in advance, and a control unit 11 for reading and executing the program stored in the program memory 10. good. The program may be stored in the program memory 10 in advance, or may be recorded on a recording medium and stored in the program memory 10 using an installation means.
The first electrode 52 and the second electrode 62 can be made of a known conductive metal such as aluminum, copper, platinum, gold, silver, titanium, or the like. The first electrode 52 and the second electrode 62 are formed to sandwich the through hole 4, and transport ions in the test liquid by applying a direct current. Therefore, the first electrode 52 only needs to be formed at a location in contact with the test liquid in the first chamber 5, such as on the surface of the substrate 3, on the inner surface of the first chamber member 51, or in the space within the first chamber 5. It may be arranged via the lead 53. Like the first electrode 52, the second electrode 62 may also be formed at a location in contact with the test liquid in the second chamber 6, such as on the surface of the substrate 3, the inner surface of the second chamber member 61, or the second chamber member 61. 6 through the lead 63. In the example shown in FIG. 1A, the first electrode 52 is formed on the inner surface of the first chamber member 51, and the second electrode 62 is formed on the inner surface of the second chamber member 61. The electrode 62 may be inserted through holes formed in the first chamber member 51 and the second chamber member 61.
The first electrode 52 is connected to a power source 54 and a ground 55 via a lead 53. The second electrode 62 is connected to the ammeter 7 and ground 64 via a lead 63. Note that in the example shown in FIG. 1B, the power source 54 is connected to the first electrode 52 side and the ammeter 7 is connected to the second electrode 62 side, but the power source 54 and ammeter 7 may be connected to the same electrode side. good.
The power source 54 is not particularly limited as long as it can supply direct current to the first electrode 52 and the second electrode 62. The ammeter 7 is not particularly limited as long as it can measure the ionic current generated over time when the first electrode 52 and the second electrode 62 are energized. Although not shown in FIG. 1B, a noise removal circuit, a voltage stabilization circuit, etc. may be provided as necessary.
When the test biological material passes through the through hole 4 of the test biological material detection device 1, the ionic current flowing through the through hole 4 is blocked by the test biological material, and the ionic current decreases. The amount of decrease in this ionic current is proportional to the volume of the biological substance to be tested within the through hole 4.
The analysis unit 8 analyzes the value (waveform) of the ion current measured by the ammeter 7.
The display section 9 only needs to be able to display the measured ion current value (waveform) and the results analyzed by the analysis section 8, and may be a known display device such as a liquid crystal display, a plasma display, or an organic EL display.

FIG. 2 shows a schematic cross-sectional view of another example of the detection device.
As shown in FIG. 2, the detection device 100 includes a first channel (first chamber) 111 formed in the first layer 110 and a second channel (second chamber) formed in the second layer 120. ) 121, a through hole 130 connecting the first flow path 111 and the second flow path 121, a first electrode 112 arranged in the first flow path 111, and a through hole 130 arranged in the second flow path 121. It has a second electrode 122.
The first flow path 111 and the second flow path 121 have introduction ports 113 and 123 for introducing the test liquid containing the biological substance 101 into the first flow path 111 and the second flow path 121, respectively. The test liquid containing the biological substance 101 to be tested is in a state where it can be distributed.
The first flow path 111 and the second flow path 121 are partitioned by a silicon substrate 140, a thin film 141 formed thereon, and a partition wall 143, and the through hole 130 is separated from the silicon substrate 140 and the thin film 141. It is formed at the center of the formed chip 142.
The first flow path 111 and the second flow path 121 may or may not be parallel, as long as they overlap in the region where the through hole 130 is formed.
In the detection device 100 having the above-described structure, a test liquid containing the biological substance to be tested 101 is introduced from the introduction ports 113 and 123 to fill the first flow path 111, the second flow path 121, and the through hole 130. After that, the biological material to be tested 101 is identified by measuring the time change in the ionic current that occurs when the biological material to be tested 101 passes through the through hole 130 .
When identifying the biological substance 101 to be tested, first, the first electrode 112 of the detection device 100 is electrically connected to the amplifier 150, the ammeter 151, and the power source 152, and the second electrode 122 of the particle identification sensor is connected to the power source. It is electrically connected to 152. Power source 152 provides a potential difference between first electrode 112 and second electrode 122 . The ammeter 151 measures the ionic current flowing between the first electrode 112 and the second electrode 122. Amplifier 150 amplifies the ion current signal.
Next, a potential is applied between the first electrode 112 and the second electrode 122 using the power source 152 (for example, a high potential is applied to the first electrode 112 and a low potential is applied to the second electrode 122). The charged biological substance 101 to be tested in the first channel 111 moves from the first channel 111 to the second channel 121 via the through hole 130 due to the potential difference. When the biological substance 101 to be tested passes through the through-hole 130, the number of ions in the test liquid in the through-hole 130 decreases, so the ionic current between the first electrode 112 and the second electrode 122 decreases. By amplifying this transient time change of the ionic current with an amplifier 150 and then measuring it with an ammeter 151, the biological substance 101 to be tested can be identified.
Identification of the biological substance to be tested 101 as described above can be performed using a biological substance to be tested detection device that includes an amplifier 150, an ammeter 151, and a power source 152. This test biological substance detection apparatus includes a housing section that can accommodate the detection device 100, a power source 152 that applies a potential between the first electrode 112 and the second electrode 122 of the detection device 100, and the first electrode 112. It includes an amplifier 150 that amplifies the ion current flowing between it and the second electrode 122, and an ammeter 151 that measures the amplified ion current. The detection device 100 may contain a test liquid containing the test biological material 101 in the first flow path 111, the second flow path 121, and the through hole 130, or may contain a test liquid containing the test biological material 101. It may be stored in a state before being filled with a test liquid, and filled with a test liquid containing the biological substance to be tested 101 at the time of measurement.
By using the test biological substance detection device having the above structure, it becomes possible to easily identify the test biological substance 101.

(Method for manufacturing detection device)
The method for manufacturing a detection device of the present invention includes coating a detection device for a biological substance to be tested with the coating composition of the present invention.
The coating method is not particularly limited, and examples thereof include a droplet dropping method, a dipping method, a roll coater method, a slit coater method, a spinner method (rotary coating method), a spray method, and the like.

Preferably, the portion of the detection device to be coated includes a portion of the detection device that is in contact with the test liquid.

Examples of the detection device before being coated with the coating composition include the above-mentioned detection device.
For example, the detection device includes a substrate in which a through hole is formed, and a first chamber and a second chamber partitioned by the substrate. The first chamber and the second chamber are communicated with each other through the through hole.
The coating is applied, for example, to at least one of the surface of the first chamber, the through hole, and the surface of the second chamber.
When detecting a test biological substance, a test liquid is provided to the first chamber, and when the test liquid provided to the first chamber passes through the through hole and moves to the second chamber, coating with a coating composition. is preferably provided on at least the surface of the first chamber and the through hole.
When detecting a biological substance to be tested, a test liquid is provided to the first chamber and the second chamber, and if the biological substance to be tested moves through the through-hole, the coating with the coating composition is applied to the surface of the first chamber and the through-hole. Preferably, it is applied to the hole and the surface of the second chamber.

(Another example of a method for detecting a biological substance to be tested)
Another example of the method for detecting a biological substance to be tested according to the present invention includes a step of passing the biological substance to be tested through a through hole formed in a detection device.
The passing step includes providing the test liquid to the through hole in the detection device manufactured by the method for manufacturing a detection device of the present invention.
An example of the passing step is performed by, for example, providing a test liquid to a first chamber and moving the test liquid from the first chamber to the second chamber through a through hole.
Another example of the passing step is performed by, for example, providing a test liquid to the first chamber and the second chamber, and moving the test biological substance from the first chamber to the second chamber through a through hole. .
The test liquid here may be the test liquid of the present invention, or unlike the test liquid of the present invention, it may not contain a nonionic surfactant. That is, the test solution here contains at least the specimen used for detecting the biological substance to be tested, and may also contain a nonionic surfactant, or may contain a nonionic surfactant. You don't have to.
An example of the test liquid here can be obtained, for example, by mixing a specimen and an aqueous solution. Specific examples and preferred examples of the specimen include, for example, the specific examples and preferred examples of the specimen mentioned in the description of the diluent of the present invention. Specific examples and preferred examples of the aqueous solution include, for example, the specific examples and preferred examples of the aqueous solution mentioned in the description of the diluent of the present invention.

(detection device)
Moreover, an example of the detection device of the present invention includes a coating film.
The coating film is a film for improving the detection sensitivity of the biological substance to be tested.
The coating film contains a nonionic surfactant.
Specific examples and preferred examples of the nonionic surfactant include, for example, the specific examples and preferred examples of the nonionic surfactant exemplified in the explanation of the diluent of the present invention.
The coating film can be obtained, for example, by coating with the coating composition of the present invention.
The detection device of the present invention including a coating film can be obtained, for example, by the method for manufacturing a detection device of the present invention.

An example of a detection device includes a substrate in which a through hole is formed, and a first chamber and a second chamber partitioned by the substrate, and the first chamber and the second chamber are communicated with each other by the through hole.
The coating film is formed, for example, at a location that comes into contact with the test liquid.
For example, the coating film is formed in the through hole.
For example, the coating film is formed on the surface of the first chamber.
For example, the coating film is formed on the surface of the second chamber.

Next, the content of the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto.

The details of the abbreviations listed below in this example are as follows.
<Phosphate buffered saline>
・D-PBS (-): Fujifilm Wako Pure Chemical Industries, Ltd., 045-29795, phosphate buffered saline ・PBS: SIGMA-ALDRICH, Phosphate buffered saline, 79383-1L diluted 10 times with pure water and prepared.

<Nonionic surfactant>
・OLFINE EXP. 4200: Nissin Chemical Industry Co., Ltd., acetylene glycol surfactant, corresponding to the above formula (2) (R ¹ and R ⁴ are isobutyl groups, R ² and R ³ are methyl groups, R ⁵ and R ⁶ are mainly It is presumed that methyl groups and hydrogen atoms are also included.)
・ADEKA Pluronic L-72: manufactured by ADEKA Co., Ltd., polyalkylene glycol surfactant, corresponding to the above formula (2-1) (R ¹ is a hydrogen atom)
・ADEKA Pluronic P-101: manufactured by ADEKA Co., Ltd., polyalkylene glycol surfactant, corresponding to the above formula (2-1) (R ¹ is a hydrogen atom)
・ADEKA Pluronic P-103: manufactured by ADEKA Co., Ltd., polyalkylene glycol surfactant, corresponding to the above formula (2-1) (R ¹ is a hydrogen atom)
・ADEKA Pluronic L-31: manufactured by ADEKA Co., Ltd., polyalkylene glycol surfactant, corresponding to the above formula (2-1) (R ¹ is a hydrogen atom)
・ADEKA Pluronic L-61: manufactured by ADEKA Co., Ltd., polyalkylene glycol surfactant, corresponding to the above formula (2-1) (R ¹ is a hydrogen atom)
・ADEKA Pluronic L-44: manufactured by ADEKA Co., Ltd., polyalkylene glycol surfactant, corresponding to the above formula (2-1) (R ¹ is a hydrogen atom)
・ADEKA Pluronic L-64: manufactured by ADEKA Co., Ltd., polyalkylene glycol surfactant, corresponding to the above formula (2-1) (R ¹ is a hydrogen atom)

<Virus>
GenomONETM-CF: Manufactured by Ishihara Sangyo Co., Ltd., CF-004

(Example 1-1-1: Preparation of diluted solution 1-1)
OLFINE EXP. 4200 was adjusted to 0.2 w/v% with D-PBS(-) to obtain diluted solution 1-1.

(Example 1-1-2: Preparation of diluted solution 1-2)
OLFINE EXP. 4200 was adjusted to 0.072 w/w% with PBS to obtain diluted solution 1-2.

(Example 1-2-1: Preparation of diluent 2-1)
OLFINE EXP. 4200 was adjusted to 0.1 w/w% with PBS to obtain diluted solution 2-1.

(Example 1-2-2: Preparation of diluent 2-2)
OLFINE EXP. 4200 was prepared with pure water to a concentration of 0.1 w/w% to obtain diluted solution 2-2.

(Example 1-3-1: Preparation of diluted solution 3-1)
Adeka Pluronic L-72 was adjusted to 0.1 w/w% with PBS to obtain diluted solution 3-1.

(Example 1-3-2: Preparation of diluted solution 3-2)
Adeka Pluronic L-72 was prepared with pure water to a concentration of 0.1 w/w % to obtain diluted liquid 3-2.

(Example 1-4-2: Preparation of diluent 4-2)
Adeka Pluronic L-31 was prepared with pure water to a concentration of 0.1 w/w % to obtain diluted liquid 4-2.

(Example 1-5-2: Preparation of diluent 5-2)
Adeka Pluronic L-61 was prepared with pure water to a concentration of 0.1 w/w % to obtain diluted solution 5-2.

(Example 1-6-2: Preparation of diluent 6-2)
Adeka Pluronic L-44 was prepared with pure water to a concentration of 0.1 w/w % to obtain diluted liquid 6-2.

(Example 1-7-2: Preparation of diluent 7-2)
Adeka Pluronic L-64 was adjusted to 0.1 w/w% with pure water to obtain diluted liquid 7-2.

(Example 1-8-2: Preparation of diluent 8-2)
Adeka Pluronic P-103 was prepared with pure water to a concentration of 0.1 w/w % to obtain diluted liquid 8-2.

(Preparation example 1: Preparation of mock test solution 1)
GenomONE ^™ -CF was used as the Sendai virus. 260 μL of HVJ-E Suspending Buffer was added to the GenomONE ^™ -CF Freeze-dried HVJ-E and suspended. Subsequently, it was diluted 500 times (volume ratio) with D-PBS(-) to obtain a mock specimen.
The obtained simulated specimen and the diluted liquid 1-1 obtained in Example 1-1-1 were mixed at a ratio of 1:1 (volume ratio) to obtain a simulated test liquid 1.

(Preparation example 2: Preparation of mock test solution 2)
GenomONE ^™ -CF was used as the Sendai virus. 260 μL of HVJ-E Suspending Buffer was added to the GenomONE ^™ -CF Freeze-dried HVJ-E and suspended. Subsequently, it was diluted 1000 times (volume ratio) with D-PBS(-) to obtain mock test solution 2.

(Preparation example 3: Preparation of mock test solution 3)
E. coli. E. coli DH5α Competent Cells (Takara Bio Inc., 9057) were used. E. coli was cultured in LB medium with shaking at 100 rpm, 37° C., for 15 hours, and then centrifuged at 7000 rpm, 4° C., for 10 minutes, to collect E. coli pellets. E. coli pellets were diluted and suspended in D-PBS (-) supplemented with 8% glycerol (Fuji Film Wako Pure Chemical Industries, Ltd., 075-00616) so that the turbidity at 590 nm was 0.1, and a mock sample was prepared. Obtained.
The obtained simulated specimen and the diluted solution 1-2 obtained in Example 1-1-2 were mixed at a ratio of 1:1 (volume ratio) to obtain a simulated test solution 3.

(Example A: Module coating example 1)
Adeka Pluronic P-101 was dissolved in a mixture of pure water and ethanol at a ratio of 1:1 (weight ratio) so that the solid content was 0.1 wt% to obtain coating liquid 1.
The module was coated with the coating liquid 1 by filling the flow path of an ipore sensor module (CSTEC Corporation, AIP003-01A) with a pore diameter of 300 nm with the obtained coating liquid 1.

(Example B: Module coating example 2)
Adeka Pluronic P-103 was dissolved in a mixed solution of pure water and ethanol at a ratio of 1:1 (weight ratio) so that the solid content was 0.1 wt% to obtain coating solution 2.
The module was coated with the coating liquid 2 by filling the flow path of an ipore sensor module (CSTEC Corporation, AIP003-01A) with a pore diameter of 300 nm with the obtained coating liquid 2.

(Example C: Module coating example 3)
Adeka Pluronic P-101 was dissolved in a mixed solution of pure water and ethanol at a ratio of 1:1 (weight ratio) so that the solid content was 0.02 wt% to obtain coating solution 3.
The module was coated with the coating liquid 3 by filling the flow path of an ipore sensor module (CSTEC Corporation, AIP003-01B) with a pore diameter of 300 nm with the obtained coating liquid 3.

(Example D: Module coating example 4)
Adeka Pluronic P-103 was dissolved in a mixed solution of pure water and ethanol in a ratio of 1:1 (weight ratio) so that the solid content was 0.05 wt% to obtain coating solution 4.
The module was coated with the coating liquid 4 by filling the flow path of an ipore sensor module (CSTEC Corporation, AIP003-01B) with a pore diameter of 300 nm with the obtained coating liquid 4.

(Example 2-1)
Fill the flow channel of an ipore sensor module (Asahi Rubber Co., Ltd., M-AS-300-A002-002-Pm) with a pore diameter of 300 nm with the simulated test solution 1 obtained in Preparation Example 1 using a pipetman. It dripped until. It was confirmed whether the dropped simulated test liquid 1 automatically entered the flow path and conducted in the measuring device. At that point, continuity was confirmed.
The device used was a particle measuring device nanoSCOUTER ^TM (Advantest Co., Ltd., WEL1100). The measurement conditions were set as Hardware Setting Range: Range 1 (user 2), MOVING Average: 1/1, and Voltage: -0.1V.
After confirming continuity, the number of detected pulses of the sample was subsequently measured. Measurement was carried out for 5 minutes under the same measurement conditions as above, and the measurement data was analyzed using Ipore PC software, pulse extraction was performed for extremely small pulses, and the number of pulses detected in 5 minutes was counted.
Table 1 shows the number of pulse measurements and the number of pulses per measurement.

(Example 2-2 and Example 2-3)
Example 2-1 except that the type of Eyepore sensor module and the number of pulse measurements were changed to the Eyepore sensor module (both have a pore diameter of 300 nm) and the number of pulse measurements listed in Table 1. Pulse measurement was performed in the same manner as in 2-1.
The results are shown in Table 1.

(Example 2-4)
As the eyepore sensor module, the module prepared in Example C and coated with coating liquid 3 was used. The number of pulses was measured three times. Other than that, pulse measurement was performed in the same manner as in Example 2-1.
The results are shown in Table 1.

(Example 2-5)
As the eyepore sensor module, the module prepared in Example D and coated with coating liquid 4 was used. The number of pulses was measured three times. Other than that, pulse measurement was performed in the same manner as in Example 2-1.
The results are shown in Table 1.

(Example 2-6)
As the eyepore sensor module, the module prepared in Example A and coated with coating liquid 1 was used. The number of times the pulse number was measured was one. Mock test liquid 2 was used as the mock test liquid. Other than that, pulse measurement was performed in the same manner as in Example 2-1.
The results are shown in Table 1.

(Example 2-7)
As the eyepore sensor module, the module prepared in Example B and coated with coating liquid 2 was used. The number of times the pulse number was measured was one. Mock test liquid 2 was used as the mock test liquid. Other than that, pulse measurement was performed in the same manner as in Example 2-1.
The results are shown in Table 1.

(Example 2-8)
The simulated test liquid 3 obtained in Preparation Example 3 was dropped into a micropore device (Advantest Co., Ltd.) with a pore diameter of 3 μm using a pipetman or the like until the channel was filled.
The micropore device was set in a particle measuring device microSCOUTER ^TM (Advantest Co., Ltd., WEL1200). The measurement conditions of microSCOUTER ^TM were set as Hardware Setting Range: Range 1 (±10 μA), MOVING Average: 1/1, Voltage: -0.1 V, and the measurement was performed for 5 minutes, and the number of E. coli bacteria detected in 5 minutes. was counted.
Table 1 shows the number of pulse measurements and the number of pulses per measurement.

(Comparative example 1)
The flow path of an ipore sensor module (Asahi Rubber Co., Ltd., M-AS-300-A002-002-Pm) with a pore diameter of 300 nm is filled with the simulated test liquid 2 obtained in Preparation Example 2 using a pipetman. It dripped until. It was confirmed whether the dropped simulated test liquid 2 automatically entered the flow path and conducted in the measuring device. However, continuity was not confirmed. Therefore, pulse measurement could not be performed.

比較例１に示すように、非イオン性界面活性剤を含有するコート液又は希釈液を用いない場合、試験液がモジュール中の微細流路に入らないためパルス測定を行うことができなかった。
それに対して、非イオン性界面活性剤を含有するコート液及び／又は希釈液を用いた実施例２－１～２－８では、パルスが測定された。

As shown in Comparative Example 1, when a coating liquid or diluting liquid containing a nonionic surfactant was not used, pulse measurement could not be performed because the test liquid did not enter the microchannel in the module.
In contrast, pulses were measured in Examples 2-1 to 2-8 in which a coating liquid and/or diluting liquid containing a nonionic surfactant were used.

(Defoaming test/conditions)
If the test liquid diluted with the diluent has air bubbles and the air bubbles block the through-hole, the biological substance to be tested will not be able to pass through the through-hole. On the other hand, if the diluent is less likely to contain bubbles, the biological substance to be tested will more easily pass through the through-hole. Therefore, the antifoaming properties of the diluted liquid were evaluated.
20 mL of each diluted solution was placed in a 30 cc (diameter 2.5 cm x height 6.5 cm) container. The container was shaken for 1 minute at 2000 rpm using a vortex mixer (Fisher Scientific). Thereafter, the time taken until the bubbles disappear was visually checked, and when the time until the bubbles disappeared was less than 1 minute, it was rated "○", and when it was 1 minute or more, it was rated "x".

(Protein adsorption test)
If the biological substance to be tested is adsorbed to the surface of the detection device before reaching the through-hole, the accuracy of detection of the biological substance to be tested decreases. Therefore, we conducted an adsorption test for the biological substance to be tested (protein in this example) on the main materials used in the detection device.

<Protein adsorption test/condition 1>
Goat anti-mouse IgG antibody HRP conjugate (manufactured by Southern Biotechnology Associates) was diluted with PBS to a concentration of 1 mg/g to prepare an IgG-HRP solution. This IgG-HRP solution and the above diluted solution were mixed at a ratio of 1:1 (volume ratio). Further, as a negative control, IgG-HRP solution and PBS were mixed at a ratio of 1:1 (volume ratio). 100 μl of each of these mixed solutions was added to 5 wells of a 96-well plate whose bottom surface was made of hydrophobic PET (Lumirror Film T-60, Toray Industries, Inc.). After standing at room temperature for 30 minutes, the mixed solution in the well was drained. Subsequently, 200 μl of PBS was added to each well and the operation of draining was repeated three times. Subsequently, 100 μl of TMB solution (Sera Care, SureBlue) was added to each well, and 1 minute later, 100 μl of STOP solution (Sera Care) was added. The absorbance at 450 nm and 650 nm was measured using a microplate reader (TECAN, infinite M200PRO). The value was calculated by subtracting the absorbance at 650 nm from the absorbance at 450 nm, and the average absorbance of 5 wells to which each mixture was added was obtained. The average absorbance in the negative control wells was taken as a protein adsorption rate of 100%, and the protein adsorption rate of the wells to which each mixture was added was calculated.

<Protein adsorption test/conditions 2>
Goat anti-mouse IgG antibody HRP conjugate (manufactured by Southern Biotechnology Associates) was diluted with PBS to a concentration of 1 mg/g to prepare an IgG-HRP solution. This IgG-HRP solution and the above diluted solution were mixed at a ratio of 1:1 (volume ratio). Further, as a negative control, IgG-HRP solution and PBS were mixed at a ratio of 1:1 (volume ratio). 100 μl of each of these mixed solutions was added to 5 wells of a 96-well plate whose bottom surface was made of a hydrophilic polyester film (product number: 9984, As One Corporation). After standing at room temperature for 30 minutes, the mixed solution in the well was drained. Subsequently, 200 μl of PBS was added to each well and the operation of draining was repeated three times. Subsequently, 100 μl of TMB solution (Sera Care, SureBlue) was added to each well, and 1 minute later, 100 μl of STOP solution (Sera Care) was added. The absorbance at 450 nm and 650 nm was measured using a microplate reader (TECAN, infinite M200PRO). The value was calculated by subtracting the absorbance at 650 nm from the absorbance at 450 nm, and the average absorbance of 5 wells to which each mixture was added was obtained. The average absorbance in the negative control wells was taken as a protein adsorption rate of 100%, and the protein adsorption rate of the wells to which each mixture was added was calculated.

<Protein adsorption test/condition 3>
Goat anti-mouse IgG antibody HRP conjugate (manufactured by Southern Biotechnology Associates) was diluted with PBS to a concentration of 1 mg/g to prepare an IgG-HRP solution. This IgG-HRP solution and the above diluted solution were mixed at a ratio of 1:1 (volume ratio). Further, as a negative control, IgG-HRP solution and PBS were mixed at a ratio of 1:1 (volume ratio). 100 μl of each of these mixed solutions was added to 5 wells of a 96-well plate whose bottom surface was made of adhesive transfer tape (9969, As One Corporation). After standing at room temperature for 30 minutes, the mixed solution in the well was drained. Subsequently, 200 μl of PBS was added to each well and the operation of draining was repeated three times. Subsequently, 100 μl of TMB solution (Sera Care, SureBlue) was added to each well, and 1 minute later, 100 μl of STOP solution (Sera Care) was added. The absorbance at 450 nm and 650 nm was measured using a microplate reader (TECAN, infinite M200PRO). The value was calculated by subtracting the absorbance at 650 nm from the absorbance at 450 nm, and the average absorbance of 5 wells to which each mixture was added was obtained. The average absorbance in the negative control wells was taken as a protein adsorption rate of 100%, and the protein adsorption rate of the wells to which each mixture was added was calculated.

(Contact angle measurement/condition 1)
4 μl of the above diluted solution was applied to a PET substrate (Lumirror Film T-60, Toray Industries, Inc.) using a pipette. The contact angle of the droplet 5 seconds after attachment was measured using a contact angle meter (Kyowa Interface Science Co., Ltd., DM-701).

(Contact angle measurement/condition 2)
4 μl of the above diluted solution was applied to the SiN substrate using a pipette. The contact angle of the droplet 1 second after attachment was measured using a contact angle meter (Kyowa Interface Science Co., Ltd., DM-701).

(Surface tension measurement)
The above diluted solution was added to the petri dish. Using a surface tension meter (Kyowa Interface Science Co., Ltd., DY-700), the surface tension was measured after 1 minute using the Willhelmy method.

(Capillary force calculation)
When liquid passes through minute channels and through holes, the smaller the capillary force, the slower the liquid passes through. When the speed is low, the liquid is less likely to generate bubbles. As a result, air bubbles can be prevented from blocking the through-hole, and the biological substance to be tested can easily pass through the through-hole.

<Capillary force calculation/condition 1>
Using the obtained surface tension γ and contact angle θ, the capillary force in the rectangular microchannel can be calculated from the following formula (F1) as described in the literature (Journal of the Japan Society for Precision Engineering Vol. 82, No. 5, 2016). Here, the capillary force of each diluted liquid was calculated with the size of the rectangular microchannel made of PET (polyethylene terephthalate) as long side ω = 0.001 m and short side d = 0.00054 m.

<Capillary force calculation/condition 2>
Using the obtained surface tension γ and contact angle θ, the capillary force in the circular microchannel can be calculated from the following formula (F2) as described in the literature (Journal of the Japan Society for Precision Engineering Vol. 82, No. 5, 2016). Here, the capillary force of each diluted liquid was calculated by setting the radius of the SiN circular microchannel (for example, through hole) to radius r = 0.00000015 m.

(Example 3-2-1)
Diluted liquid 2-1 was subjected to the antifoaming test and protein adsorption test described above. The results are shown in Table 2.

(Example 3-2-2)
The above contact angle measurement and surface tension measurement were performed on diluted liquid 2-2. The results are shown in Table 3.
Further, the capillary force under each of Conditions 1 and 2 was calculated using the obtained contact angle and surface tension. The results are shown in Table 3.

(Example 3-3-1)
Diluted liquid 3-1 was subjected to the antifoaming test and protein adsorption test described above. The results are shown in Table 2.

(Example 3-3-2)
Regarding diluted liquid 3-2, the above-mentioned contact angle measurement and surface tension measurement were performed. The results are shown in Table 3.
Further, the capillary force under each of Conditions 1 and 2 was calculated using the obtained contact angle and surface tension. The results are shown in Table 3.

(Comparative example 2-1-1)
Table 2 shows the results of the defoaming test for PBS. The negative controls for the above protein adsorption tests 1 to 3 are shown in Table 2 as Comparative Example 2-1-1.

From Table 2, it was found that when the diluted solutions of Examples were used, the amount of protein adsorbed was small in the protein adsorption test. That is, the diluted liquid has the ability to suppress the adsorption of the biological substance to be tested, and is considered to improve the detection sensitivity of the detection device for the biological substance to be tested. Further, it was found that the diluted liquid had sufficient antifoaming properties, and the flow path was not easily blocked by air bubbles in the diluted liquid.

なお、「Ｅ＋」は１０のべき乗を表す。例えば、「Ｅ＋０５」は、１０^５を表し、－１.５５Ｅ＋０５は、－１．５５×１０^５を表す。表４も同様である。

Note that "E+" represents a power of 10. For example, "E+05" represents 10 ⁵ , and -1.55E+05 represents -1.55×10 ⁵ . The same applies to Table 4.

From Table 3, it was found that when the diluted solutions of Examples were used, the contact angle and surface tension were low. That is, it is considered that the wettability is high and the flow path is not likely to be clogged.

(Surface tension measurement)
The above diluted solution was added to the petri dish. Using a surface tension meter (Kyowa Interface Science Co., Ltd., DY-700), the surface tension was measured after 1 minute using the Willhelmy method.

(Contact angle measurement/condition 3)
4 μl of the above diluted solution was applied to the silicone rubber sheet using a pipette. The contact angle of the droplet 1 second after attachment was measured using a contact angle meter (Kyowa Interface Science Co., Ltd., DM-701).

<Capillary force calculation/condition 3>
Using the obtained surface tension γ and contact angle θ, the capillary force in the circular microchannel can be calculated from the following formula (F2) as described in the literature (Journal of the Japan Society for Precision Engineering Vol. 82, No. 5, 2016). Here, the capillary force of each diluted liquid was calculated by setting the radius of the circular microchannel made of silicone rubber (for example, a silicone tube) to be radius r = 0.00025 m.

(Capillary force confirmation test)
When the capillary force calculated above is negative, it indicates that the liquid flows through the capillary tube, and when it is positive, it indicates that the liquid does not flow through the capillary tube. With respect to the calculation results of the capillary force calculated under Condition 3, we verified whether the same results would be obtained using an actual capillary.

<Passage test of diluted liquid>
A silicon tube with an inner diameter of 0.5 mm (radius of 0.00025 m) was held vertically and immersed 1 cm from the lower end into the diluted solution prepared above. Thereafter, the silicone tube was pulled up from the diluent and the height of the diluted liquid level, which had risen due to capillary force, was confirmed. When the height of the liquid level was greater than 1 cm, it was evaluated as "○", when it was 1 cm or less and greater than 0.1 cm, it was evaluated as "Δ", and when it was 0.1 cm or less, it was evaluated as "x".

(Example 4-2-2)
For diluted liquid 2-2, contact angle measurement and diluted liquid passage test were conducted under the above condition 3. The results are shown in Table 4.
In addition, the above-mentioned surface tension measurement was carried out. The results are shown in Table 4.
Furthermore, the capillary force under Condition 3 was calculated using the obtained contact angle and surface tension. The results are shown in Table 4.

(Examples 4-3-2 to 4-8-2 and Comparative Example 4-1-2)
For the diluted solutions shown in Table 4 below, contact angle measurements and diluted solution passage tests were conducted under Condition 3 above. The results are shown in Table 4.
In addition, the above-mentioned surface tension measurement was carried out. The results are shown in Table 4.
Furthermore, the capillary force under Condition 3 was calculated using the obtained contact angle and surface tension. The results are shown in Table 4.

In a diluted liquid passage test, in a diluted liquid on which capillary force acts, the liquid level becomes higher than the outside of the tube by the amount of capillary force. As shown in Table 4, for the diluted liquids of Examples, the liquid level of all the diluted liquids for which the capillary force calculation results were negative increased significantly, and even for the diluted liquids for which the capillary force was positive, the liquid level was lower than that of water. The surface was rising. Based on the above, we believe that we have verified that the calculation results of capillary force were correct.

1 Test biological substance detection device 2 Detection device 3 Substrate 4 Through hole 5 First chamber 6 Second chamber 7 Ammeter 8 Analyzer 9 Display unit 10 Program memory 11 Control unit 41 Test biological substance 51 First chamber member 52 First electrode 53 Lead 54 Power source 55 Ground 61 Second chamber member 62 Second electrode 63 Lead 64 Ground 101 Test biological substance 100 Detection device 110 First layer 111 First channel 112 First electrode 113 Inlet 120 Second layer 121 Second channel 122 Second electrode 123 Inlet 130 Through hole 140 Silicon substrate 141 Thin film 142 Chip 143 Partition 150 Amplifier 151 Ammeter 152 Power supply

Claims (19)

A diluent for diluting a specimen used for detection of a biological substance to be tested, which contains a nonionic surfactant that improves permeability of the biological substance to be tested into a through-hole in a detection device. , a diluent in which the nonionic surfactant is at least one of an acetylene glycol surfactant and a polyalkylene glycol surfactant. A diluent for diluting a specimen used for detection of a biological substance to be tested, which contains a nonionic surfactant that improves permeability of the biological substance to be tested into a through-hole in a detection device. , a diluent in which the nonionic surfactant is represented by the following formula (1).
A ¹ -B-A ² ...Formula (1)
(In formula (1), A ¹ and A ² each independently represent a monovalent group exhibiting hydrophilicity, and B represents a divalent group exhibiting relative hydrophobicity compared to A ¹ and A ² . (Represents an organic group.) The diluent according to claim 1 or 2, wherein the capillary force given by the following formula (F1) or (F2) in the microchannel is negative.

(γ in formulas (F1) and (F2) represents the surface tension of the diluted liquid, and θ represents the contact angle of the diluted liquid. Also, d in formula (F1) is the short side of the rectangular flow path, ω represents the long side of the rectangular flow path, and r in equation (F2) represents the radius of the circular flow path.) The diluent according to claim 3, wherein the inner surface of the microchannel is made of resin, ceramics, semimetal, or metal. The diluent according to claim 1 or 2, further comprising an aqueous solution. 6. The diluent according to claim 5, wherein the aqueous solution is selected from physiological saline and phosphate buffered saline. The diluent according to claim 1 or 2, wherein the biological substance to be tested is at least one of bacteria, viruses, nucleic acids, extracellular vesicles, and proteins. The diluent according to claim 1 or 2, wherein the detection of the test biological material is performed by the test biological material passing through the through hole formed in the detection device. A test liquid containing a specimen used for detecting a test biological substance and a nonionic surfactant that improves permeability of the test biological substance to a through hole in a detection device, the test liquid comprising: A test solution in which the ionic surfactant is at least one of an acetylene glycol surfactant and a polyalkylene glycol surfactant. A test liquid containing a specimen used for detecting a test biological substance and a nonionic surfactant that improves permeability of the test biological substance to a through hole in a detection device, the test liquid comprising: A test liquid in which the ionic surfactant is represented by the following formula (1).
A ¹ -B-A ² ...Formula (1)
(In formula (1), A ¹ and A ² each independently represent a monovalent group exhibiting hydrophilicity, and B represents a divalent group exhibiting relative hydrophobicity compared to A ¹ and A ² . (Represents an organic group.) The test liquid according to claim 9 or 10, wherein the capillary force given by the following formula (F1) or (F2) in the microchannel is negative.

(γ in formulas (F1) and (F2) represents the surface tension of the test liquid, and θ represents the contact angle of the test liquid. Also, d in formula (F1) is the short side of the rectangular flow path, ω represents the long side of the rectangular flow path, and r in equation (F2) represents the radius of the circular flow path.) The test liquid according to claim 9 or 10, wherein the detection of the test biological material is performed by the test biological material passing through the through hole formed in the detection device. A method for preparing a test liquid, comprising mixing a specimen used for detecting a biological substance to be tested and the diluent according to claim 1 or 2. A coating composition for improving the detection sensitivity of a test biological substance, the composition comprising a nonionic surfactant that improves the permeability of the test biological substance to a through-hole in a detection device, A coating composition, wherein the nonionic surfactant is at least one of an acetylene glycol surfactant and a polyalkylene glycol surfactant. A coating composition for improving the detection sensitivity of a test biological substance, the composition comprising a nonionic surfactant that improves the permeability of the test biological substance to a through-hole in a detection device, A coating composition in which the nonionic surfactant is represented by the following formula (1).
A ¹ -B-A ² ...Formula (1)
(In formula (1), A ¹ and A ² each independently represent a monovalent group exhibiting hydrophilicity, and B represents a divalent group exhibiting relative hydrophobicity compared to A ¹ and A ² . (Represents an organic group.) A diluent containing a nonionic surfactant that improves the permeability of biological substances passing through the flow path. The diluent according to claim 16, wherein the nonionic surfactant is represented by the following formula (1).
A ¹ -B-A ² ...Formula (1)
(In formula (1), A ¹ and A ² each independently represent a monovalent group exhibiting hydrophilicity, and B represents a divalent group exhibiting relative hydrophobicity compared to A ¹ and A ² . (Represents an organic group.) A coating composition that suppresses the adhesion of biological substances within a flow path, the coating composition containing a nonionic surfactant. The coating composition according to claim 18, wherein the nonionic surfactant is represented by the following formula (1).
A ¹ -B-A ² ...Formula (1)
(In formula (1), A ¹ and A ² each independently represent a monovalent group exhibiting hydrophilicity, and B represents a divalent group exhibiting relative hydrophobicity compared to A ¹ and A ² . (Represents an organic group.)

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