JP5253137B2 - Target substance detection element and method for manufacturing target substance detection element - Google Patents

Description

  The present invention relates to a target substance detection element for detecting a target substance in a specimen and a method for manufacturing the target substance detection element.

  Patent Documents 1 and 2 disclose a sensor using plasmon resonance as a target substance detection element capable of detecting a target substance in a specimen in real time and in a label-free manner.

On the other hand, Patent Document 3 discloses a target substance detection element in which an organic linker layer is formed on the surface of a metal film and a polymer layer for further fixing an antigen is formed thereon for the purpose of fixing the antigen to the sensor substrate. It is disclosed.
Special registration 03452837 JP-A-63-75542 JP 2000-146976 A

  However, in order to improve the sensitivity of the plasmon resonance sensor described in Patent Documents 1 and 2, even if the linker layer and the polymer layer described in Patent Document 3 are formed on the surface of the sensor, the layers of the linker layer and the polymer layer are formed. Therefore, the distance between the target substance capturing body and the sensor surface is increased, which causes a problem that highly sensitive detection cannot be performed.

  Therefore, the present invention provides a target substance detection element that can be detected with high sensitivity and a method for manufacturing the same.

The target substance detection element of the present invention has a linker layer provided on a metal surface, and a target substance capturing body bonded to the linker layer,
The linker layer is composed of a chain polymer, and the polymer includes a repeating unit in which a first side chain is bonded to the surface of the metal and a second side chain is bonded to the target substance capturing body. It is the polymer which has.

Here, the polymer has a repeating unit A for binding to the surface of the metal on the first side chain, and a repeating unit B for binding to the target substance capturing body on the second side chain; A polymer having
Or a polymer comprising a repeating unit C having a site that binds to the surface of the metal on the first side chain and a site that binds to the target substance capturing body on the second side chain,
It is preferable that

The method for producing a target substance detection element of the present invention comprises:
Forming a linker layer on the surface of the metal;
Binding a target substance capturing body to the linker layer, and forming the linker layer on the surface of the metal,
A chain-shaped polymer, wherein the polymer has a first side chain bonded to the surface of the metal, and a second side chain bonded to the target substance capturing body; And a step of bringing the metal into contact with a solution having a pH lower than the pH at which the polymer associates.

One embodiment of the present invention
A target substance detection element,
A linker layer provided on a portion made of gold or silver on the surface of the metal structure;
A target substance capturing body bonded to the linker layer,
The linker layer comprises a non-aggregate of a chain polymer having no cross-linked structure,
The polymer is
A polymer comprising a repeating unit A having an amino group in the side chain and a repeating unit B having a carboxyl group in the side chain, or a polymer comprising a repeating unit C having an amino group and a carboxyl group in the side chain. Target substance detection element.

Another embodiment of the present invention is as follows.
A method of manufacturing a target substance detection element,
Forming a linker layer on the gold or silver portion of the surface of the metal structure;
Binding a target substance capturing body to the linker layer, and forming a linker layer on the surface of the metal structure,
A polymer having no cross-linked structure, comprising a repeating unit A having an amino group in the side chain and a repeating unit B having a carboxyl group in the side chain, or a repeating having an amino group and a carboxyl group in the side chain A method for producing a target substance detection element, comprising a step of bringing a metal structure into contact with a solution having a pH lower than the pH at which the polymer associates, the polymer comprising a unit C.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to obtain the target substance detection element which can detect a target substance with high sensitivity, and its manufacturing method.

  Hereinafter, the present invention will be described in detail.

The first of the present invention is
A method of manufacturing a target substance detection element,
Forming a linker layer on the gold or silver portion of the surface of the metal structure;
Binding a target substance capturing body to the linker layer, and forming a linker layer on the surface of the metal structure,
A polymer having no cross-linked structure, comprising a repeating unit A having an amino group in the side chain and a repeating unit B having a carboxyl group in the side chain, or a repeating having an amino group and a carboxyl group in the side chain A method for producing a target substance detection element, comprising the step of bringing the metal structure into contact with a solution having a pH lower than the pH at which the polymer associates, the polymer comprising a unit C.

  Hereinafter, the first steps of the present invention will be described with reference to FIG.

<Step of forming a linker layer on the surface of the metal structure>
FIG. 2 (i) shows a step of forming a linker layer on the surface of the metal structure, 3 shows the metal structure, and 5 shows the linker layer.

  The metal structure 3 may be a metal structure that has a portion made of gold or silver on the surface and can generate a plasmon resonance phenomenon, and the shape is not limited. Examples of the shape of the metal structure include a thin film shape, a fine particle shape, a ring shape, and a cubic shape.

  When detecting using localized surface plasmon resonance, the metal structure 3 may have a fine particle shape or a pattern shape.

  When the detection is performed using localized surface plasmon resonance and the metal structure 3 is a fine particle, the diameter of the metal structure 3 is preferably 10 nm to 500 nm. In addition, when the metal structure 3 has a pattern shape other than fine particles such as a ring shape or a cubic shape, the size of the metal structure 3 is preferably 10 nm to 1450 nm, and more preferably 50 nm to 450 nm. Here, the size of the metal structure 3 is the maximum value of the distance between any two points on the plane of the metal structure parallel to the surface on which the metal structure 3 is formed.

  Moreover, when detecting using surface plasmon resonance, it is preferable that the metal structure 3 is a thin film shape, and it is preferable that the thickness of the metal structure 3 is 10 nm-100 nm. By setting the thickness of the metal structure 3 within these ranges, surface plasmon resonance capable of achieving the target detection sensitivity can be effectively generated.

  The metal structure 3 may be made entirely of gold or silver as long as it has a surface containing gold or silver and can generate plasmon resonance. The metal structure 3 may be made of gold or silver and other materials. May be included. When the metal structure 3 has a portion containing gold or silver and a portion made of other material, the portion made of the other material is a metal that causes a plasmon resonance phenomenon other than gold and silver, for example, , Copper, aluminum, or an alloy thereof.

  As shown in FIG. 3 (I), the metal structure 3 may be present by itself, or the metal structure 3 may be a base as shown in FIG. 1 (II). 4 may be formed on the surface. When the metal structure 3 exists by itself, the metal structure 3 functions as a support for the target substance detection element.

  When the metal structure 3 is formed on the surface of the substrate 4, the material of the substrate 4 can be a glass substrate, a quartz substrate, a resin substrate such as polycarbonate or polystyrene, an ITO substrate, or the like. However, when a signal is detected using the plasmon resonance phenomenon, that is, when light absorption is detected as transmitted light, any material can be used as long as it has a level of transparency that can be detected by light absorption. It is. In addition, the base | substrate 4 may be comprised by one layer and may be comprised by the some layer. When the substrate 4 is composed of a plurality of layers, the outermost layer is preferably a layer made of chromium, titanium, or the like. By making the outermost layer a layer made of chromium, titanium, or the like, the adhesion between the substrate 4 and the metal structure 3 is improved.

  When a plurality of metal structures 3 are formed on the surface of the substrate 4, as shown in FIG. 4, the metal structures 3 may be regularly provided in a 1 mm to 10 mm square region to form an array. preferable. In the case of an array, the interval between the metal structures 3 is preferably in the range of 50 nm to 2 μm, more preferably 150 nm to 1 μm. If the interval is too narrow, the plasmons of the metal structures interact with each other, affecting the spatial electric field distribution / intensity, which may reduce the sensor sensitivity. On the other hand, if the interval is too wide, the density of the metal structure is low, so that the signal intensity becomes weak and a special optical system may be required. As an example, FIG. 12 shows an SEM image of a ring-shaped metal structure formed in an array on the surface of the substrate.

  In the case where the plurality of metal structures 3 are formed on the surface of the base 4, for example, they can be formed by a method as shown in FIG. 5.

  Specifically, the method is as follows.

  A metal film 20 is formed on the surface of the substrate 4 (FIG. 5A) by sputtering or vapor deposition (FIG. 5B). Then, an electron beam resist film 21 is formed on the surface of the metal film 20 by a method such as spin coating (FIG. 5C). Only a portion of the electron beam resist film 21 where the metal structure is to be formed is exposed with an electron beam drawing apparatus and developed to obtain a resist pattern 22 (FIG. 5D). Thereafter, an unnecessary metal film is etched (FIG. 5E), and the resist pattern 22 is removed to obtain a metal structure 3 arranged in an array (FIG. 5F). Here, as an apparatus for exposing the resist, in addition to an electron beam drawing apparatus, a focused ion beam processing apparatus, an X-ray exposure apparatus, an EUV exposure apparatus, an excimer laser exposure apparatus, and the like can also be used.

  Moreover, as shown in FIG. 6, it is also possible to produce the metal structure 3 using the fine uneven base 4 (FIG. 6 (1)) produced by a molding method. In this case, the metal film 20 is formed on the uneven surface of the substrate 4 by a method such as sputtering or vapor deposition (FIG. 6B). Next, the metal structure 20 formed on the surface of the substrate is obtained by polishing so that the metal film 20 remains only in the concave portion of the substrate (FIG. 6 (3)).

  When the metal film 20 is thinner than the unevenness of the substrate 4, the state (3) is obtained without going through the state (2) in FIG. 6, and a metal structure can be obtained. In this case, the height of the convex portion of the base body 4 may be higher than the height of the metal structure 3. Further, the metal structure 3 may be formed on the wall surface of the uneven portion of the base body 4. Instead of polishing, the metal film 20 can be removed by using etch back by dry etching.

  Next, a method for forming the linker layer 5 on the surface of the metal structure 3 will be described.

  The method of forming the linker layer 5 on the surface of the metal structure 3 is a polymer having no cross-linked structure, which is a repeating unit A having an amino group in the side chain and a repeating unit B having a carboxyl group in the side chain. A polymer comprising a repeating unit C having an amino group as a functional group and a carboxyl group as a functional group in a side chain, and a solution having a pH lower than the pH at which the polymer is associated with the metal structure This can be done by bringing the body into contact.

  The linker layer 5 is a polymer comprising a repeating unit A having an amino group in the side chain and a repeating unit B having a carboxyl group in the side chain, or a polymer comprising a repeating unit C having an amino group and a carboxyl group in the side chain. Therefore, the ratio of amino groups and carboxyl groups contained in the polymer is high. Therefore, since many amino groups of the linker layer are bonded to a portion containing gold or silver existing on the surface of the metal structure, the linker layer is firmly fixed to the metal structure. The amino group is generally known to be bonded to gold and silver by ammonia or a group obtained by removing hydrogen from a primary or secondary amine (Japanese Patent No. 3452837, http: //Www.molsci.jp/discussion_past/2003/BK2003/Abs/3/3Cp07.pdf). Further, a large number of target substance capturing bodies can be bound to the linker layer by the large number of carboxyl groups of the linker layer. Furthermore, since the polymer does not have a cross-linked structure, it is possible to form a thin linker layer. Here, the crosslinked structure means that the side chains of the linear (chain) polymer are bonded by a covalent bond.

Examples of the polymer comprising the repeating unit A having an amino group in the side chain and the repeating unit B having a carboxyl group in the side chain include diallylamine, N-vinylformamide, N-vinylacetamide, N-vinylpropionamide, N -Vinylbutyramide, N-vinylvaleramide, N-isopropenylformamide, N-isopropenylacetamide, N-isopropenylpropionamide, N-isopropenylbutyramide, N-isopropenylvaleramide, lysine, arginine, histidine, etc. And a structure containing an amino group (corresponding to the repeating unit A) and a carboxylic acid such as maleic acid, succinic acid, acrylic acid, methacrylic acid, crotonic acid, cinnamic acid, itaconic acid, fumaric acid, glutamic acid, aspartic acid Structure (in repeating unit B Copolymers of a combination of a person) can be mentioned. Among these, a copolymer of diallylamine and maleic acid is preferable, and a polymer represented by the general formula (1) is more preferable.
General formula (1)

(L: m = 0.004 to 13, and l and m are integers of 1 or more. Further, the molecular weight of the copolymer represented by the general formula (1) is 1000 to 200000.)

  Examples of the polymer composed of repeating units C having an amino group and a carboxyl group in the side chain include diallylamine, N-vinylformamide, N-vinylacetamide, N-vinylpropionamide, N-vinylbutyramide, N- In structures containing amino groups such as vinyl barrelamide, N-isopropenylformamide, N-isopropenylacetamide, N-isopropenylpropionamide, N-isopropenylbutyramide, N-isopropenylvaleramide, lysine, arginine, histidine, etc. An amino group was introduced into a structure containing a carboxylic acid such as a derivative introduced with a carboxyl group, maleic acid, succinic acid, acrylic acid, methacrylic acid, crotonic acid, cinnamic acid, itaconic acid, fumaric acid, glutamic acid or aspartic acid. Derivative, or above Such derivatives obtained by binding a structure comprising a structure and a carboxyl group containing an amino group.

  When a metal structure is brought into contact with a solution containing these polymers and having a pH lower than the pH at which the polymers are associated, protonation to the amino group of the polymer occurs, and the entire molecule has a charge. The proportion of polymer increases. That is, the proportion of the polymer that becomes difficult to associate due to electrostatic repulsion increases. Thereby, a polymer is fixed to the surface of a metal structure, without laminating | stacking.

  On the other hand, when the pH of the solution is the pH at which the polymer associates, the ratio of the polymer having no charge as a whole molecule increases, and the polymers associate and are stacked and fixed on the surface of the metal structure. It is thought. Further, it is considered that the amount of the linker layer immobilized varies due to the association.

  In addition, when the pH of the solution is higher than the pH at which the polymer associates, the ratio of the polymer having a charge as a whole molecule is high and the polymers are difficult to associate with each other, but the prototyping to the amino group of the polymer is not possible. Nation is unlikely to occur, so the bond with the gold or silver portion of the polymer will be poor, and it will be difficult to fix the polymer.

  It is also possible to prepare the solution after cutting the crosslinked structure of the polymer having a crosslinked structure by reduction or the like. Further, when the polymer does not have a crosslinked structure, but interaction between hydrophobic groups of the polymer occurs, it is preferable to include a dispersant in the solution containing the polymer.

  In a sensor using plasmon resonance, a signal is generated in response to a change in the dielectric constant in the vicinity of the surface of the metal structure due to the target substance binding to the target substance detection element, and the place where the change in the dielectric constant occurs is the location of the metal structure. The closer to the surface, the greater the signal. Therefore, by setting the pH of the solution containing the polymer to a pH lower than the pH at which the polymer associates, it is possible to fix the polymer to the surface of the metal structure without laminating the target substance capturing body. It can be fixed closer to the surface of the metal structure. Thereby, a highly sensitive target substance detection element is obtained.

  In the present invention, the pH at which the polymer associates refers to the pH at the highest absorbance and the lowest pH at 660 nm obtained when the pH of the polymer solution of 0.25% is changed to 2-9. It is defined as a pH having an absorbance higher than 1/100 of the highest absorbance in a spectrum having a difference from the absorbance of 0.5 or more.

  Next, as shown in FIG. 2 (ii), the target substance capturing body 1 is bound to the formed linker layer 5.

  The target substance capturing body 1 for capturing the target substance is bound to the carboxyl group of the polymer constituting the linker layer 5 formed. As a method of binding the carboxyl group of the linker layer and the target substance capturing body, a method of binding the carboxyl group of the linker layer and the amino group of the target substance capturing body by a dehydration condensation reaction or the like can be used. When an amino group does not exist in the target substance capturing body, it can be bonded to the linker layer by introducing and derivatizing the amino group through a spacer or the like. The dehydration condensation reaction can be performed by, for example, dropping an aqueous solution containing a target substance capturing body and a coupling reagent such as 1-Ethyl-3- [3-dimethylamino] propyl] carbohydrate hydride into the linker layer.

  Examples of such a combination of the target substance capturing body 1 and the target substance 2 include antigen-antibody, enzyme-substrate, DNA-DNA and the like.

  In addition to the linker layer 5, a nonspecific adsorption preventing layer may be formed on the surface of the metal structure. By forming such a non-specific adsorption preventing layer, non-specific adsorption of impurities on the surface of the metal structure can be reduced. As a material constituting such a nonspecific adsorption preventing layer, skim milk, casein, bovine serum albumin, phospholipid, polyethylene glycol, and derivatives thereof are preferably used. In addition, when forming a nonspecific adsorption | suction prevention layer, it is desirable to form after making a target substance capture body couple | bond with a linker layer.

By such a method, the target substance detection element of the present invention,
A linker layer provided on a portion made of gold or silver on the surface of the metal structure;
A target substance capturing body bonded to the linker layer,
The linker layer comprises a non-aggregate of a chain polymer having no cross-linked structure,
The polymer is
A polymer comprising a repeating unit A having an amino group in the side chain and a repeating unit B having a carboxyl group in the side chain, or a polymer comprising a repeating unit C having an amino group and a carboxyl group in the side chain. Target substance detecting element can be obtained. The repeating unit A having an amino group and a carboxyl group in the side chain may be a repeating unit having an amino group and a carboxyl group in the same side chain, or has a plurality of side chains, and the plurality of side chains Of these, repeating units each having an amino group and a carboxyl group in different side chains may be used.

  FIG. 1 shows an example of the target substance detection element of the present invention. The target substance 2 detection element according to an embodiment of the present invention is a linker layer provided on the surface of a metal (metal structure 3). 5 and the target substance capturing body 1 bound to the linker layer. The linker layer is made of a chain polymer, and the polymer has a repeating unit in which the first side chain is bonded to the surface of the metal and the second side chain is bonded to the target substance capturing body. It is a coalescence.

  Here, the polymer has a repeating unit A for binding to the surface of the metal on the first side chain, and a repeating unit B for binding to the target substance capturing body on the second side chain. It can be.

  Alternatively, it may be a polymer comprising a repeating unit C having a site that binds to the metal surface on the first side chain and a site that binds to the target substance capturing body on the second side chain.

  The method for producing a target substance detection element includes a step of forming a linker layer on a metal surface and a step of binding a target substance capturing body to the linker layer.

  The step of forming the linker layer on the metal surface is a chain polymer, and the polymer has a first side chain bonded to the metal surface and a second side chain. A step of contacting the metal with a solution containing a polymer having a site for binding to the target substance capturing body and having a pH lower than the pH at which the polymer is associated.

  In the present invention, the non-aggregate of the polymer means a polymer composed of an unassociated polymer.

  The target substance detection element (10 in FIG. 7) obtained by the present invention can be bonded to the substrate 11 having the reaction well 6, the flow path 7, the inlet 8, and the outlet 9 to form a target substance detection substrate. When the target substance detection element 10 and the substrate 11 are bonded together, the surface of the substrate 11 on which the reaction well 6 and the flow path 7 are formed and the metal structure of the base 4 of the target substance detection element 10 are formed. The target surface can be used as a target substance detection substrate having a shape as shown in FIG. In FIG. 7, 12 is a light source, 14 is a light detection device, and 19 is a collimating lens. Here, the reaction well 6 forms a region (reaction mechanism) for bringing the target substance detection element into contact with the specimen.

  When the substrate 4 has the function of the substrate 11, the substrate 4 preferably has a reaction well, a flow channel, an inlet, and an outlet. However, the flow channel, the inlet, and the outlet are formed by cells that come into contact with the substrate. It may be a thing.

The substrate 11 having the reaction well 7 and the flow channel 10 on the surface can be detected by light absorption when detecting a signal using the plasmon resonance phenomenon, that is, when detecting light absorption as transmitted light, like the base 4. A material having a degree of permeability is preferable. Specifically, it is preferable to produce by a plate made of polydimethylsiloxane (PDMS) used in a so-called μTAS (Micro Total Analysis System) type apparatus.
In addition, a microtiter plate or μTAS chip is packaged in a moisture-proof and light-shielding bag filled with inert gas, and a sample dilution reagent containing BSA (bovine serum albumin), a reagent for washing after reaction, and before reaction A special reagent for reference spectrum measurement and the like can be bundled and provided as a target substance detection kit.

  Next, an example of a target substance detection method using the target substance detection element obtained by the present invention will be described with reference to FIG.

  In FIG. 8, 6 is a reaction well, 7 is a flow path, 8 is an inlet, 9 is an outlet, 10 is a target substance detection element, 13 is a light source unit, 14 is a light detection device, 15 is a liquid feed pump, and 16 is a waste liquid reservoir. , 17 is a central processing unit, and 18 is a display unit.

  A specimen (solution) containing a target substance is filled into the reaction well 6 via the inlet 8 and the flow path 7 using the liquid feed pump 15, and the specimen and the target substance detection element 10 are brought into contact with each other to perform incubation for a certain period of time. When the target substance specifically binds to the target substance capturing body provided on the surface of the metal structure of the target substance detection element 10, the plasmon resonance state on the surface of the metal structure changes.

  In such a state, the target substance detection element 10 is irradiated with light generated from the optical unit 13, and the emitted light emitted from the target substance detection element 10 is acquired by the light detection device 14 as a signal, so that the plasmon resonance state is obtained. Can be detected as a detection signal. The emitted light from the target substance detection element 10 measured by the light detection device 14 may be reflected light or transmitted light.

  Then, the result measured by the photodetection device 14 is compared and calculated by the central processing unit 17 with calibration data prepared in advance using a target substance-containing solution having a known concentration, the concentration of the target substance is specified, the concentration, etc. Is displayed on the display unit 18.

  The reaction well 6 and the flow path 7 are polydimethyl used in an open polystyrene well typified by a 96-well or 384-well microtiter plate, or a so-called μTAS (Micro Total Analysis System) type apparatus. It can be made with a siloxane (PDMS) substrate. Moreover, as the liquid feed pump 15, a micro piston pump, a syringe pump, etc. are used.

  Hereinafter, the present invention will be described more specifically with reference to examples. In addition, this invention is not limited only to a following example.

(Example 1, Comparative Example 1, Comparative Example 2)
First, PAS-410 is used as a polymer comprising a repeating unit A having an amino group as a functional group in the side chain and a repeating unit B having a carboxyl group as a functional group in the side chain, and the pH at which the polymer associates is determined. It was measured.

The absorbance of a solution in which PAS-410 was dispersed in a phosphate buffer at a concentration of 0.25% was measured. The obtained absorbance spectrum is shown in FIG. The structure of PAS-410 is shown in the following general formula (2).
General formula (2)

(J: k = 1: 1, j, k = 44 to 90)

  From FIG. 17 (a), the absorbance of the solution containing the polymer at pH 3.6 is the maximum value of 2.018, and at pH 2.0, the absorbance is the minimum value of -0.001, and the maximum value and the minimum value are It can be seen that the difference is 0.5 or more, and the absorbance is greater than 0.020 at pH 3.2 to 4.6.

  Therefore, the polymer begins to associate from pH 3.2 and associates to pH 4.6. That is, the pH at which the polymer associates was found to be pH 3.2 to 4.6.

  Next, a target substance detection element was produced. FIG. 9 shows the structure of the target substance detection element used. In FIG. 9, 1 is a target substance capturing body, 3 is a metal structure, 5 is a linker layer, 6 is a reaction well, 12 is a light source, and 14 is a light detection device. The reaction well 6 functions as a base of the target substance detection element. Further, a tungsten lamp was used as the light source 12 and a spectrophotometer was used as the light detection device 14.

  An amination microtiter plate (manufactured by Corning), which is a reaction well 6, is immersed in a solution containing gold colloid, which is a metal structure 3 having a particle size of 100 nm (manufactured by British Biocell), for 12 hours. The colloidal gold was dispersed and fixed on the bottom of the substrate. FIG. 10 shows a scanning electron microscope (SEM) image of the fixed gold colloid. In addition, it was confirmed that the absorption spectrum of colloidal gold has a peak wavelength in the vicinity of 530 nm.

  Next, the linker layer 5 was formed on the surface of the gold colloid produced above.

  A solution containing PAS-410 (manufactured by Nitto Boseki Co., Ltd.) adjusted to a concentration of 0.25% and pH 2 to 9 was dropped on the surface of the gold colloid with a spotter to form a linker layer on the surface of the metal structure. After washing, 1-Ethyl-3- [3-dimethylamino] propyl] carbohydride hydrochloride (manufactured by Sigma Aldrich) is an aqueous solution and a target substance capturing body (+)-Biotin- (PEO) 4-Amine (Molecular Bioscience) (Product made) A mixed solution of an aqueous solution was put in a well and reacted at 37 ° C. for 1 hour. After the completion, it was further immersed in a 0.1% bovine serum albumin phosphate buffer for 2 hours to produce a target substance detection element.

Next, the following operation was performed.
(1) A phosphate buffer solution of 100 μg / ml rabbit anti-Biotin antibody as a sample was introduced into the prepared target substance detection element with a pipette and reacted at 37 ° C. for 2 hours, and then the rabbit anti-Biotin antibody was (+) -Combined with Biotin- (PEO) 4-Amine.
(2) The specimen in the well was discharged, a phosphate buffer was introduced with a pipette, and the inside of the reaction well was washed.
(3) Finally, a phosphate buffer was introduced into the well, and the absorption spectrum of the target substance detection element was measured.

  As shown in FIG. 9, the absorption spectrum of the target substance detection element is obtained by irradiating the target substance detection element with the light generated from the light source 12 and detecting the transmitted light from the target substance detection element using the light detection device 14. . FIG. 17B is a graph in which the shift amount of the obtained absorption spectrum is on the vertical axis and the pH of the solution containing PAS-410 at the time of preparing the target substance detection element is on the horizontal axis.

  In addition, the case where the solution of PAS-410 is less than pH 3.2 is Example 1, the case where pH is 3.2 to 4.6 is Comparative Example 1, and the case where pH is greater than 4.6. It is comparative example 2.

  In Example 1, the absorbance is 0.020 or less as shown in FIG. 17A, and it can be seen that the peak shift amount of the target substance detection element is large as shown in FIG. 17B. This is thought to be due to protonation of the amino group of the polymer, repulsion of the polymers due to electrostatic repulsion, and fixation of the polymer to the surface of the metal structure without stacking.

  In Comparative Example 2, the absorbance is 0.020 or less as shown in (a) of FIG. 17 and it is considered that no association has occurred. However, as shown in (b) of FIG. The peak shift amount of the element is small. As described above, this is because it becomes difficult for the polymer to be fixed to a portion made of gold or silver on the surface of the metal structure because the protonation to the amino group of the polymer is less likely to occur. it is conceivable that. In addition, since the polymer is difficult to be fixed, it is considered that the peak shift amount becomes smaller due to peeling of the polymer when washing is performed a plurality of times.

  Furthermore, in Comparative Example 1, as shown in FIG. 17A, it can be seen that the absorbance is greater than 0.020 and the polymers are associated. Among them, when the pH is 3.2 to 3.6, the absorbance increases as the pH increases. In addition, as the pH increases, the peak shift amount of the target substance detection element decreases. Therefore, when the pH of the solution containing the polymer is 3.2 to 3.6, as the pH increases, the proportion of the associated polymer increases, and the polymer is laminated on the surface of the metal structure. It is considered that pH 3.6 is a maximum value and has reached the peak of the association. Further, when the pH is 3.6 to 4.6, the peak shift of the target substance detection element is slightly increased as the pH is increased as shown in FIG. This is more strongly affected at pH 3.6 to 4.6 due to the lower proportion of polymer associating with increasing pH than increasing non-protonated amino groups. It is thought that there is.

(Example 2)
In forming the linker layer, the solution of pH 2 was used instead of the solution of pH 2 to 9, and after binding the target substance capturing body to the linker layer, 2% in 1% bovine serum albumin phosphate buffer was used. In addition to soaking for a period of time, a target substance detection element was produced in the same manner as in Example 1 except that it was soaked in 0.025% skim milk buffer for 2 hours.

  Comparing the obtained absorption spectrum before and after the reaction, as shown in FIG. 11, the target substance binds to the surface of the target substance detection element by a specific antigen-antibody reaction, so that the absorption spectrum moves to the longer wavelength side. Shifted. In addition, since the correlation between the peak intensity of the absorption spectrum or the shift amount of the peak wavelength and the anti-Biotin antibody concentration in the sample is obtained in advance with a known anti-Biotin antibody control solution, the anti-Biotin antibody of the sample of unknown concentration can be obtained by the above operation. It is also possible to determine the concentration.

(Comparative Example 3, Comparative Example 4)
In Comparative Example 3 and Comparative Example 4, the amount of spectrum shift of the target substance detection element when a linker layer different from that in Example 1 is used is shown.

  As the linker layer, bovine serum albumin, which is a polymer having a crosslinked structure, was used in Comparative Example 3, carboxymethyldextran having no repeating unit A having an amino group was used in Comparative Example 4, and Comparative Example 3 And the target substance detection element was produced by the method similar to Example 2 except having used the solution of pH 7.6 when forming a linker layer in Comparative Example 4.

  1 mg / ml rabbit anti-Biotin antibody phosphate buffer as a sample was introduced into the wells of the target substance detection element prepared in Comparative Example 3 and Comparative Example 4, and reacted at 37 ° C. for 2 hours, followed by target substance detection. The shift amount of the absorption spectrum of the device was obtained.

  The shift amounts of the absorption spectra of Example 2, Comparative Example 3, and Comparative Example 4 are shown in FIG. The amount of shift of the absorption spectrum when using the target substance detection element of Example 2 was 3.2 times for Comparative Example 3 and 1.6 times for Comparative Example 4.

(Example 3)
In this example, it is shown that not only purified samples as in Examples 1 and 2 but also crude human serum can be detected by the target substance detection element obtained by the present invention.

  In this example, in the target substance detection element of Example 2, the target substance detection element was obtained in the same manner as in Example 2 except that an aminated blood group A antigen (manufactured by Dextra Laboratories) was used as the target substance capturing body. Was made.

  Sample 1 and sample 2 used in this example are human sera collected and separated from persons with different O types, and both anti-blood type A antibodies and anti-blood type B antibodies are contained in human sera. It was a thing.

Next, the following measurement operation was performed.
(1) A phosphate buffer solution containing 0.25% PAS-410, 0.01% BSA, and 0.01% skim milk is used as a reagent for sample dilution, and the volume ratio of the sample and the sample dilution reagent is 1: 2. The sample was diluted by mixing and stirring.
(2) The specimen diluted by the above method was introduced into the well of the prepared target substance detection element by a pipette, and the anti-blood type A antibody was captured by the blood type A antigen possessed by the target substance detection element.
(3) The sample solution in the well was discharged, a phosphate buffer solution was introduced with a pipette, and the inside of the reaction well was washed.

  The shift amount of the absorption spectrum of the gold colloid before and after the reaction is shown in FIGS. Specimen 1 has 0.4 times the noise due to nonspecific adsorption and 1.0 times the amount of signal due to specific binding reaction compared to the case where no reagent dilution reagent is used. The noise due to adsorption was 0.5 times, and the signal amount due to specific binding reaction was 1.5 times.

  Therefore, crude human serum can also be measured, and when the specimen is diluted with the specimen dilution solution, it is more preferable than when diluted with a phosphate buffer, and the noise due to non-specific adsorption is reduced or equivalent, It was found that the signal amount due to a specific binding reaction can be equivalent or increased.

Example 4
In this example, it is shown that quantitative analysis is possible with the target substance detection element obtained by the present invention.

  In this example, in the detection apparatus used in Example 2, an aminated blood group A antigen (manufactured by CARBOHYDRATE SYNTHSIS) was immobilized as a target substance capturing body by the same operation as Example 2, and 0.01% bovine It was immersed in serum albumin phosphate buffer for 2 hours and then immersed in 0.025% skim milk buffer for 2 hours to obtain a target substance detection element. In addition, an aminated blood group B antigen (CARBOHYDRATE SYNTHSIS) was immobilized by the same operation to obtain an anti-blood type B antibody detection element.

  Prepare specimen prepared by diluting specimen 1 (same specimen as in Example 3) 2 times and 8 times with phosphate buffer, and implement with anti-blood type A antibody detection element and anti-blood type B antibody detection element Using the specimen dilution reagent having the same composition as in Example 2, anti-blood type A antibody and anti-blood B antibody contained in the specimen were measured by the same measurement procedure as in Example 2.

  The results are shown in FIG. Here, (i) is data obtained by measuring anti-blood type A antibody, and (ii) is data obtained by measuring anti-blood type B antibody.

  From these results, it is understood that a signal change corresponding to the dilution factor occurs, and the anti-blood type A antibody and the anti-blood type B antibody contained in the specimen can be quantitatively detected.

It is a schematic diagram which shows an example of the target substance detection element of this invention. It is a schematic diagram showing an example of a metal structure, a linker layer, and a target substance detection element. It is a schematic diagram which shows the state of an example of a metal structure. It is a schematic diagram which shows an example of the array-shaped metal structure formed in the base | substrate surface. It is a schematic diagram which shows an example of the method of forming a metal structure on a base surface. It is a schematic diagram which shows an example of the method of forming a metal structure on a base surface. It is a schematic diagram which shows an example of a target substance detection apparatus. It is a schematic diagram which shows an example of a target substance detection apparatus. 1 is a schematic diagram showing a target substance detection device of Example 1. FIG. 2 is an SEM image of a metal structure formed on a substrate surface in Example 1. FIG. 10 is a diagram showing a change in optical characteristics of a target substance detection element before and after contact with a specimen in Example 2. It is a SEM image of the metal structure formed in the array form on the substrate surface. It is a figure which shows the signal of the target substance detection element of Example 2, the comparative example 3, and the comparative example 4. FIG. 4 is data showing noise due to non-specific adsorption when using a phosphate buffer and a reagent dedicated for specimen dilution in Example 3. FIG. 4 is a data showing a signal when using a phosphate buffer and a reagent for reagent dilution in Example 3. FIG. In Example 4, (i) data obtained by measuring anti-blood type A antibody contained in specimen 1 (ii) data obtained by measuring the amount of anti-blood group B antibody contained in specimen 1. (A) Data indicating the relationship between absorbance at 660 nm and pH of 0.25% PAS-410, (b) Formation of a linker layer using a solution containing 0.25% PAS-410 at pH 2.0 to 9.0 It is the data which shows the signal of the target substance detection element when doing.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Target substance capture body 2 Target substance 3 Metal structure 4 Base body 5 Linker layer 6 Reaction well 7 Flow path 8 Inlet 9 Outlet 10 Target substance detection element 11 Substrate 12 Light source 13 Light source unit 14 Photodetection device 15 Liquid feed pump 16 Waste liquid reservoir 17 Central processing unit 18 Display unit 19 Collimate lens 20 Metal film 21 Electron beam resist film 22 Resist pattern

Claims (6)

A target substance detection element,
A linker layer provided on a portion made of gold or silver on the surface of the metal structure;
A target substance capturing body bonded to the linker layer,
The linker layer comprises a non-aggregate of a chain polymer having no cross-linked structure,
The polymer is
A polymer comprising a repeating unit A having an amino group in the side chain and a repeating unit B having a carboxyl group in the side chain, or a polymer comprising a repeating unit C having an amino group and a carboxyl group in the side chain. Target substance detection element.   The target substance detection element according to claim 1, wherein the polymer is a copolymer of diallylamine and maleic acid. The target substance detection element according to claim 2, wherein the copolymer of diallylamine and maleic acid is a polymer represented by the following general formula (1).
General formula (1)

(L: m = 1: 0.0004 to 13, and l and m are integers of 1 or more. The molecular weight of the copolymer represented by the general formula (1) is 1000 to 200000.) A method of manufacturing a target substance detection element,
Forming a linker layer on the gold or silver portion of the surface of the metal structure;
Binding a target substance capturing body to the linker layer, and forming a linker layer on the surface of the metal structure,
A chain polymer not having a cross-linked structure, comprising a repeating unit A having an amino group in the side chain and a repeating unit B having a carboxyl group in the side chain, or an amino group and a carboxyl group in the side chain A target substance detection element comprising a step of contacting the metal structure with a solution having a pH lower than the pH at which the polymer associates, the polymer comprising a repeating unit C having Method.   The method for producing a target substance detection element according to claim 4, wherein the polymer is a copolymer of diallylamine and maleic acid. The method for producing a target substance detection element according to claim 5, wherein the copolymer of diallylamine and maleic acid is a polymer represented by the following general formula (1).
General formula (1)

(L: m = 1: 0.0004 to 13, and l and m are integers of 1 or more. The molecular weight of the copolymer represented by the general formula (1) is 1000 to 200000.)