WO2014021171A1

WO2014021171A1 - Sensor member fabrication method, sensor chip fabrication method, and sensor member usage method

Info

Publication number: WO2014021171A1
Application number: PCT/JP2013/070073
Authority: WO
Inventors: 幸登中村; 正貴松尾
Original assignee: コニカミノルタ株式会社
Priority date: 2012-07-30
Filing date: 2013-07-24
Publication date: 2014-02-06
Also published as: JPWO2014021171A1

Abstract

[Problem] To provide a sensor member fabrication method, a sensor chip fabrication method, and a sensor member usage method, whereby, even when mass producing sensor chips, easy production is possible, production costs can be significantly reduced, and it is possible to suppress variations in detection precision owing to individual differences. [Solution] A fabrication method for a sensor member which is employed in an optical-type specimen detection device which carries out a detection of a specimen by an illumination of an excitation light upon a metallic film which is formed upon a dielectric member comprises: a step of forming a metallic film upon a translucent flexible substrate; a step of forming a solid phase film upon the metallic film formed upon the flexible substrate, said solid phase film being employed to anchor the specimen; and a step of cutting the flexible substrate whereupon the metallic film and the solid phase film are formed to a prescribed size,.

Description

Method for manufacturing sensor member, method for manufacturing sensor chip, and method for using sensor member

The present invention relates to a surface plasmon resonance (SPR) measuring device and a surface based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS; Surface Plasmon-field enhanced Fluorescence Spectroscopy) applying the surface plasmon resonance phenomenon. The present invention relates to a sensor chip used in an optical analyte detection device such as a plasmon excitation enhanced fluorescence measurement device, a method for manufacturing a sensor member used in the sensor chip, and a method for using the sensor member.

2. Description of the Related Art Conventionally, when detecting a very small substance, various specimen detection apparatuses that can detect such a substance by applying a physical phenomenon of the substance have been used.
As one of such analyte detection devices, we apply the phenomenon (SPR: Surface Plasmon Resonance (SPR) phenomenon) in which high light output is obtained by resonating electrons and light in a minute region such as the nanometer level. For example, a surface plasmon resonance device (hereinafter referred to as “SPR device”) that detects minute analytes in a living body can be used.

In addition, based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) using the surface plasmon resonance (SPR) phenomenon, analyte detection can be performed with higher accuracy than the SPR device. The surface plasmon excitation enhanced fluorescence spectrometer (hereinafter referred to as “SPFS device”) is also one of such specimen detection devices.

In this surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), surface plasmon light is applied to the surface of the metal thin film under the condition that excitation light such as laser light emitted from a light source attenuates total reflection (ATR) on the surface of the metal thin film. By generating (dense wave), the amount of photons contained in the excitation light irradiated from the light source is increased to several tens to several hundreds times, and the electric field enhancement effect of the surface plasmon light is obtained.

FIG. 13 is a schematic configuration diagram illustrating an example of a conventional SPFS apparatus.
The SPFS device 100 includes a sensor chip 106 including a prism-shaped dielectric member 102 having a substantially trapezoidal vertical cross-sectional shape and a metal thin film 104 formed on a horizontal upper surface 102a of the dielectric member 102. The sensor chip 106 is loaded in the sensor chip loading unit 108 of the SPFS device 100.

Further, as shown in FIG. 13, a light source 110 is arranged on one side surface 102 b below the dielectric member 102, and incident light 112 from the light source 110 is below the dielectric member 102. Then, the light enters the side surface 102b of the dielectric member 102 and is irradiated through the dielectric member 102 toward the metal thin film 104 formed on the upper surface 102a of the dielectric member 102.

On the metal thin film 104 of the sensor chip 106, a flow channel 114 for sending a sample liquid containing an analyte, which is a sample to be detected, is formed. A sensor region 116 on which a ligand that specifically binds to the analyte is immobilized is provided.

The analyte to be detected is labeled with, for example, a fluorescent substance, and the fluorescent substance is excited by the surface plasmon light (dense wave) generated by the incident light 112 irradiated from the light source 110, and the fluorescence 118 is emitted. It emits light.

Further, above the sensor chip 106, a light detection means 120 for receiving the fluorescence 118 is provided.
In the SPFS device 100 configured in this manner, the light source 110 irradiates the metal thin film 104 with incident light 112 at an incident angle α that is ATR, and the surface plasmon light (dense wave) generated on the surface of the metal thin film 104 causes an analyte Excitation of a fluorescent substance for labeling.

Then, the light 118 emitted from the excited fluorescent material is received by the light detection means 120 to measure the light quantity of the fluorescence 118, and based on the light quantity of the fluorescence 118, the ratio of the analyte in the sample liquid Is configured to ask for.

Also in the conventional SPR device, as described in Patent Document 1, the incident light 112 irradiated from the light source 110 is incident on the sensor chip 106, and the reflected light reflected on the metal thin film 104 is received by the light receiving means. By receiving the light, the intensity of the reflected light can be measured, and the ratio of the analyte in the sample liquid can be obtained based on the intensity of the reflected light.

As the sensor chip 106 used in such an SPR device or SPFS device, a metal thin film deposited on the upper surface of a glass prism (dielectric member 102) was used. However, since a glass prism is expensive, it is not practical to replace a sensor chip using such a glass prism every time an analyte is measured because the measurement cost increases.

For this reason, a sensor chip using a resin prism instead of a glass prism is also used (Patent Document 1).

JP 2007-192841 A

However, a sensor chip in which a metal thin film is directly formed on a dielectric member such as a glass prism or a resin prism, or a sensor chip in which a metal thin film is formed on the inner bottom surface of a container-like part as in Patent Document 1, the sensor Even in the case of mass production of chips, it is difficult to significantly reduce the manufacturing cost.

Further, in the sensor chip as described above, the variation in detection accuracy due to individual differences has increased, and there has been concern about the reliability of detection accuracy.

In the present invention, in view of such a current situation, even when mass-producing sensor chips, it can be easily manufactured, manufacturing costs can be greatly reduced, and variation in detection accuracy due to individual differences can be suppressed. A method for manufacturing a sensor member, a method for manufacturing a sensor chip, and a method for using the sensor member are provided.

The present invention has been invented in order to solve the above-described problems in the prior art, and in order to achieve at least one of the above-described objects, a sensor member reflecting one aspect of the present invention is provided. The manufacturing method is a method for manufacturing a sensor member used in an optical sample detection apparatus that detects a sample by irradiating excitation light onto a metal film formed on a dielectric member,
Forming a metal film on a flexible substrate having translucency;
Forming a solid phase film used for immobilizing the specimen on the metal film formed on the flexible substrate;
Cutting the flexible base material on which the metal film and the solid phase film are formed into a predetermined size.

In addition, a sensor chip manufacturing method reflecting another aspect of the present invention is used for an optical sample detection apparatus that detects a sample by irradiating a metal film formed on a dielectric member with excitation light. A method for manufacturing a sensor chip, comprising:
Fixing the sensor member manufactured by any one of the above-described sensor member manufacturing methods on the dielectric member;
A sample solution containing the sample is injected onto the dielectric member to which the sensor member is fixed, and a reaction space forming member for forming a reaction space for causing the reaction between the solid phase film and the sample is fixed. Process.

In addition, a method for using a sensor member reflecting still another aspect of the present invention includes a dielectric member, and an optical specimen detection device having a light source disposed on one side surface below the dielectric member. A method of using a sensor member used in
Attaching a sensor member manufactured by any one of the above-described sensor member manufacturing methods on the dielectric member;
Irradiating the metal film of the sensor member with excitation light from the light source;
It is characterized by having.

According to the present invention, a metal thin film and a solid phase film are formed on a flexible base material in advance to produce a film-like sensor member, and the film-like sensor member is fixed to a dielectric member to provide a sensor. Since the chip only needs to be manufactured, the manufacturing cost can be greatly reduced even when the sensor chip is mass-produced.

In addition, since the plurality of sensor members are manufactured by cutting the sheet-like flexible substrate, individual differences among the sensor members can be suppressed, and the reliability of detection accuracy of the sensor chip can be improved. .

FIG. 1 is a schematic configuration diagram showing the configuration of a sensor member manufactured by the sensor member manufacturing method of the present invention described below. FIG. 2 is a schematic configuration diagram for explaining the flow of the manufacturing method of the sensor member of FIG. FIG. 3 is a schematic configuration diagram for explaining the convex portions 19 a and 19 b provided on the flexible base material 18. FIG. 4 shows a configuration in which the flexible substrate 18 is wound up by the substrate take-up device 24 after the metal film 14 is formed on the flexible substrate 18 drawn out from the substrate feeding device 22. It is a schematic block diagram to explain. FIG. 5 is a schematic configuration diagram showing a configuration of a sensor chip manufactured by the method for manufacturing a sensor chip of the present invention. FIG. 6 is a schematic configuration diagram for explaining the flow of the manufacturing method of the sensor chip of the present invention. FIG. 7 is a schematic configuration diagram when the dielectric member 32 and the reaction space forming member 34 are fixed by fixing the reaction space forming member 34 and the sensor chip holding member 40 using the fastening member 42. FIG. 8 is a schematic configuration diagram showing a configuration of an optical specimen detection apparatus 50 using the sensor member 10 of the present invention. FIG. 9 is a graph showing the relationship between the incident angle of the excitation light 54 measured using the sensor member 10 manufactured by the sensor member manufacturing method of the present invention and the SPR signal. FIG. 10 is a graph showing the relationship between the incident angle of the excitation light 54 measured using the sensor member 10 manufactured by the sensor member manufacturing method of the present invention and the SPFS signal. FIG. 11 is a graph showing the relationship between the incident angle of excitation light measured using a conventional sensor chip and the SPR signal as a comparative example. FIG. 12 is a graph showing the relationship between the incident angle of excitation light measured using a conventional sensor chip and the SPFS signal as a comparative example. FIG. 13 is a schematic configuration diagram illustrating an example of a conventional SPFS apparatus.

Hereinafter, embodiments (examples) of the present invention will be described in more detail based on the drawings.
1. FIG. 1 is a schematic configuration diagram showing the configuration of a sensor member manufactured by the method for manufacturing a sensor member of the present invention shown below. FIG. 2 is a flow chart of the method for manufacturing the sensor member of FIG. It is a schematic block diagram for doing.
As shown in FIG. 1, the sensor member 10 of the present invention includes a sensor base 12 having translucency and flexibility, a metal film 14 provided on the sensor base 12, and a metal film 14 provided on the sensor base 12. And the solid phase film 16 formed.

The sensor substrate 12 is not particularly limited as long as it has translucency and flexibility, but the refractive index n of the sensor substrate 12 is at least 1.4 or more, preferably 1.5 or more. It is hoped that.

Since the sensor substrate 12 has such a refractive index n, as will be described later, the electric field enhancement can be increased and measurement can be performed with high accuracy.
The “translucency” of the sensor substrate 12 is required to transmit at least the excitation light irradiated from the light source of the SPR device or the SPFS device and to transmit light of all wavelengths, as will be described later. There is no.

Examples of such sensor substrate 12 include polyesters such as polyethylene terephthalate (PET) and polyethylene naphthalate, polyolefins such as polyethylene (PE) and polypropylene (PP), cyclic olefin copolymer (COC), and cyclic olefin polymer. Polycyclic olefins such as (COP), vinyl resins such as polyvinyl chloride and polyvinylidene chloride, polystyrene, polyetheretherketone (PEEK), polysulfone (PSF), polyethersulfone (PES), polycarbonate (PC) Polyamide, polyimide, acrylic resin, triacetyl cellulose (TAC), or the like can be used.

In particular, ZEONOR (registered trademark) manufactured by Nippon Zeon Co., Ltd. using a cyclic olefin polymer (COP) having an optical performance (such as BK7) that is comparable to that of optical glass or the like is preferable.

The material of the metal film 14 is not particularly limited, but is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, more preferably It is made of gold and may be made of an alloy of these metals.

The solid phase film 16 has a ligand for capturing an analyte immobilized when detecting an analyte using an SPR device or an SPFS device described later. -Assembled Monolayer (self-assembled monolayer) and a solid-phased layer formed on the SAM.

Examples of such a solid phase layer include glucose, carboxymethylated glucose, vinyl esters, acrylic esters, methacrylic esters, olefins, styrenes, crotonic esters, itaconic acid diesters, Including a polymer composed of at least one monomer selected from the group consisting of monomers included in maleic acid diesters, fumaric acid diesters, allyl compounds, vinyl ethers and vinyl ketones. Preferred are hydrophilic polymers such as dextran and dextran derivatives, vinyl esters, acrylic esters, methacrylic esters, olefins, styrenes, crotonic esters, itaconic diesters, maleic diesters, fumarate acid It is more preferable to include a hydrophobic polymer composed of hydrophobic monomers included in esters, allyl compounds, vinyl ethers and vinyl ketones, and dextran such as carboxymethyldextran (CMD) is biocompatible. From the viewpoints of suppressing nonspecific adsorption reaction and high hydrophilicity, it is particularly preferable.

In addition, in the stage which manufactures the sensor member 10, since the solid-phased layer is not necessarily required, it does not need to have the solid-phased layer as the solid-phase film 16.
The sensor member 10 of the present invention configured as described above is manufactured by the following process.

First, as shown in FIG. 2, a flexible substrate winding body 20 around which a long flexible substrate 18 to be a sensor substrate 12 is wound is attached to a substrate feeding device 22. The flexible substrate 18 is fed out sequentially.

Then, the metal film 14 is sequentially formed on the fed flexible base material 18 by the metal film forming means 26. The method for forming the metal film 14 is not particularly limited, and examples thereof include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. . It is preferable to use a sputtering method or a vapor deposition method because it is easy to adjust the thin film formation conditions.

Further, the thickness of the metal film 14 is not particularly limited, but preferably gold: 5 to 500 nm, silver: 5 to 500 nm, aluminum: 5 to 500 nm, copper: 5 to 500 nm, platinum: 5 Desirably within the range of ˜500 nm and their alloys: 5 to 500 nm.

From the viewpoint of the electric field enhancement effect described later, more preferable thicknesses of the metal film 14 are: gold: 20 to 70 nm, silver: 20 to 70 nm, aluminum: 10 to 50 nm, copper: 20 to 70 nm, platinum: 20 to 70 nm and their alloys: preferably in the range of 10-70 nm.

It should be noted that the upper surface shape of the metal film 14 is not limited to a planar shape, but can of course be applied to a case where the metal film 14 is formed in an uneven surface shape formed in a lattice shape, for example.

Next, the solid phase film 16 is sequentially formed on the metal film 14 by the solid phase film forming means 27. The method for forming the solid phase film 16 is not particularly limited, and a conventionally known method can be used. For example, the flexible substrate 18 is replaced with 10-carboxy-1-decanethiol (Co., Ltd.). And a method of dipping in an ethanol solution containing Dojindo Laboratories). In this way, the thiol group of 10-carboxy-1-decanethiol binds to the metal and is immobilized, and self-assembles on the surface of the metal film to form a SAM.

More preferably, it is desirable to form the solid phase film 16 only at a position necessary for the sensor member 10, that is, only in the reaction area 10 a of the sensor member 10 by inkjet coating. By forming the solid phase film 16 in this way, the use of the material for forming the solid phase film 16 can be reduced, and the manufacturing cost of the sensor member 10 can be reduced.

In the case of forming the solid phase layer, a step of forming the solid phase layer may be provided after the SAM is formed. A method for forming the solid phase layer is not particularly limited, and a conventionally known method can be used. For example, the flexible substrate 18 is composed of carboxymethyldextran (CMD) having a molecular weight of 500,000 at 1 mg / kg. Immerse in MES buffered saline (MES) (ionic strength: 10 mM) at pH 7.4 containing mL, 0.5 mM N-hydroxysuccinimide (NHS) and 1 mM water-soluble carbodiimide (WSC). Examples thereof include a method and a method performed by inkjet coating.

Next, the flexible base material 18 on which the metal film 14 and the solid phase film 16 are formed is cut into a size necessary for the sensor member 10 by the base material cutting means 28. The size required for the sensor member 10 is particularly limited as long as it can be attached to a dielectric member and can secure the reaction area 10a when a sensor chip is used, as will be described later. is not.

In addition, as a cutting method of the flexible base material 18, for example, after cutting in the traveling direction of the flexible base material 18 (hereinafter referred to as “length direction”) with a slitter knife or the like, the flexible base material 18 is flexible with a cutter or the like. The rectangular sensor member 10 can be manufactured by cutting in a direction perpendicular to the traveling direction of the substrate 18 (hereinafter referred to as “width direction”).

More preferably, the sensor member 10 having the optimum size and shape for a sensor chip to be described later is obtained by cutting (punching) the flexible base material 18 into a shape necessary for the sensor member 10 by punching. Can do.

It should be noted that the circular sensor member 10 is preferably formed by punching. Excitation light emitted from a light source used in an optical specimen detection apparatus such as an SPFS apparatus, which will be described later, is usually light having a circular irradiation shape. Can be matched. For this reason, in the sensor member 10, excitation light is not irradiated and the area | region which is not used for a measurement does not arise.

Further, even when a highly birefringent material such as polyethylene terephthalate (PET) is used as the flexible substrate 18 (sensor substrate 12), the sensor member 10 is punched along the optical axis. The use as the sensor member 10 can be prevented from being affected.

In the present embodiment, the metal film 14 forming step, the solid phase film 16 forming step, and the flexible substrate 18 cutting step are described as being performed continuously. It can also be done.

In this case, as shown in FIG. 3, it is preferable to use a flexible base material in which convex portions 19 a and 19 b are provided on both

edge portions

18 a and 18 b in the width direction of the flexible base material 18.
With this configuration, as shown in FIG. 4, the metal film 14 and the solid phase film 16 are formed on the flexible base material 18 drawn out by the base material feeding device 22, and then the base material winding is performed. It can be wound up by the take-up device 24.

The height of the protrusions 19a and 19b is such that when the flexible substrate 18 is wound up, the flexible substrate 18 does not directly touch the metal film 14 and the solid phase film 16. It may be sufficient, and can be appropriately determined according to the thickness of the metal film 14 and the thickness of the solid phase film 16.

By manufacturing sensor members in this way, even when mass-producing sensor chips, it can be easily manufactured, manufacturing costs can be greatly reduced, and variations in detection accuracy due to individual differences can be suppressed. Can do.

2. Sensor Chip Manufacturing Method The sensor member 10 manufactured in this way is used by being attached to a sensor chip used in, for example, an SPR device or an SPFS device.
FIG. 5 is a schematic configuration diagram showing a configuration of a sensor chip manufactured by the sensor chip manufacturing method of the present invention described below.

As shown in FIG. 5, the sensor chip 30 of the present invention includes a prism-shaped dielectric member 32 having a substantially trapezoidal vertical cross-sectional shape, a sensor member 10 mounted on the dielectric member 32, and an analyte. A reaction space forming member 34 for injecting a sample liquid to be included and forming a reaction space for causing a reaction between the solid phase layer of the solid phase film 16 of the sensor member 10 and the analyte in the sample liquid is formed. The

The dielectric member 32 is not particularly limited, but optically transparent, for example, various inorganic materials such as glass and ceramics, natural polymers, and synthetic polymers can be used. Chemical stability, production From the viewpoints of stability and optical transparency, those containing silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ) are preferred.

By using the same material as the sensor base material 12 of the sensor member 10 as the material of the dielectric member 32, the optical influence of the sensor base material 12 interposed between the dielectric member 32 and the metal film 14 is affected. Can be small.

In this embodiment, the prism-shaped dielectric member 32 having a substantially trapezoidal vertical cross-sectional shape is used. However, the vertical cross-sectional shape is a triangle (so-called triangular prism), a semicircular shape, a semi-elliptical shape, etc. The shape of the member 32 can be changed as appropriate.

Further, as shown in FIG. 5A, the reaction space forming member 34 may be a well member 35a capable of temporarily storing a sample liquid containing an analyte, or FIG. As shown in (), a flow path member 35b that can circulate the sample liquid to the reaction area 10a may be used.

As shown in FIG. 5A, the well member 35a constitutes a wall of the reaction area 10a so as to surround the reaction area 10a of the sensor member 10, and forms a reaction space 34a.
For example, by injecting the sample liquid into the reaction space 34 a using a pipette or the like, the analyte in the sample liquid reacts with the solid phase layer of the solid phase film 16 of the sensor member 10, and the solid phase. The analyte will be trapped in the activated layer.

On the other hand, as shown in FIG. 5B, the flow path member 35b forms a flow path 36 by the dielectric member 32 and the flow path member 35b so that the sample liquid circulates to the reaction area 10a. It is configured.

That is, the reaction space 34a is formed on the reaction area 10a of the flow path 36, and the analyte liquid in the sample liquid and the solid phase film 16 of the sensor member 10 are held by circulating the sample liquid in the reaction space 34a. The analyte is trapped in the solid phase layer by reaction with the phase phase layer.

The method for allowing the sample liquid to flow through the flow path 36 is not particularly limited, but a pump (not shown) is connected to both

end portions

36a and 36b of the flow path 36 so that the sample liquid is unidirectionally supplied. The sample liquid may be circulated to the reaction area 10a by injecting the sample liquid from the end 36a of the flow path 36 using a pipette and sucking and discharging the sample liquid with the pipette. Also good.

In particular, by reciprocating the sample liquid with respect to the reaction area 10a, the reaction efficiency between the analyte and the solid phase layer is increased even with a small amount of sample liquid, and the detection accuracy of the analyte is improved. Can do.

Further, the material of the reaction space forming member 34 (well member 35a, flow path member 35b) is not particularly limited, and for example, various inorganic materials such as glass and ceramics, natural polymers, and synthetic polymers may be used. it can.

When the reaction area 10a is covered with the reaction space forming member 34 as in the flow path member 35b, fluorescence can be observed from above the sensor chip 30 when the sensor chip 30 is used in an SPFS device, as will be described later. Thus, it is necessary to use an optically transparent material.

The sensor chip 30 of the present invention configured as described above is manufactured by the following process.
First, as shown in FIG. 6A, the sensor member 10 is fixed to the dielectric member 32 via the refractive index matching liquid 38.

The refractive index matching liquid 38 is not particularly limited, and a conventionally known refractive index matching liquid (matching oil) can be used. As the refractive index matching liquid 38, for example, by using an adhesive such as an ultraviolet curable adhesive, the refractive index matching between the dielectric member 32 and the sensor member 10 can be obtained, and the dielectric member 32 and the sensor. The member 10 can be bonded and fixed.

Note that the sensor member 10 is preferably fixed to the dielectric member 32 so that the sensor member 10 can be removed. By configuring in this way, the sensor member 10 is removed from the sensor chip 30 used for the detection of the analyte, and the sensor member 30 is manufactured by reusing the dielectric member 32 simply by attaching a new sensor member 10. can do.

For this reason, although it is excellent in an optical characteristic, even if it is a case where an expensive glass prism is utilized as the dielectric material member 32, since a glass prism can be reused, the manufacturing cost of the sensor chip 30 is reduced. be able to.

Next, as shown in FIG. 6B, the reaction space forming member 34 is placed on the dielectric member 32 to which the sensor member 10 is fixed, and the reaction space forming member 34 and the dielectric member 32 are, for example, Fix using adhesive 29 or the like.

When the sensor member 10 is configured to be removable, for example, the dielectric member 32 and the reaction space forming member 34 may be fixed by a fastening member such as a screw. In the case of fixing with a fastening member, the dielectric member 32 and the reaction space forming member 34 may be directly fastened and fixed. However, as shown in FIG. 7, the dielectric member 32 is attached to the sensor chip holding member 40. In this state, the dielectric member 32 and the sensor member 10 are sandwiched between the reaction space forming member 34 and the sensor chip holding member 40 and fixed using, for example, a fastening member 42 such as a screw. Also good.

Further, it is preferable that the seal member 44 is placed so as to surround the sensor member 10 and is fixed so that the seal member 44 is sandwiched between the dielectric member 32 and the reaction space forming member 34.

Thus, by providing the sealing member 44, the sealing property (sealing property) between the dielectric member 32 and the reaction space forming member 34 can be ensured, and when the sample liquid is injected into the reaction space 34a. The specimen liquid does not leak from between the dielectric member 32 and the reaction space forming member 34.

3. Method of Using Sensor Member FIG. 8 is a schematic configuration diagram showing a configuration of an optical specimen detection apparatus 50 using the sensor member 10 of the present invention.
As shown in FIG. 8, in the optical sample detection device 50, the sensor chip 30 having the sensor member 10 attached on the dielectric member 32 is loaded in the sensor chip loading unit 52.

Further, as shown in FIG. 8, a light source 52 is arranged on one side surface 32 b below the dielectric member 32, and excitation light 54 from the light source 52 is below the dielectric member 32. Then, the light is incident on the side surface 32b of the dielectric member 32 and irradiated through the dielectric member 32 toward the metal film 14 of the sensor member 10 attached to the upper surface of the dielectric member 32. .

As the light source 52, for example, an LD (Laser Diode), an LED (Light Emitting Diode), an HID (High Intensity Discharge) lamp (high intensity discharge lamp), or the like can be used.

Further, a polarizing filter 56 is provided between the light source 52 and the dielectric member 32 to convert the excitation light 54 emitted from the light source 52 into P-polarized light that efficiently generates surface plasmons on the metal film 14. ing.

Further, as shown in FIG. 8, light receiving means 58 for receiving the metal film reflected light 55 obtained by reflecting the excitation light 54 by the metal film 14 is provided on the other side surface 32 c below the dielectric member 32. ing.

The light source 52 is provided with incident angle adjusting means (not shown) that can appropriately change the incident angle of the excitation light 54 emitted from the light source 52 with respect to the metal film 14. On the other hand, the light receiving means 58 is also provided with a movable means (not shown). Even when the reflection angle of the metal film reflected light 55 changes, the metal film reflected light 55 is reliably received in synchronization with the light source 52. Is configured to do.

The incident angle adjusting means of the light source 52 and the moving means of the light receiving means 58 are not particularly limited. For example, the light source 52 and the light receiving means 58 are rotated using a stepping motor or a gear train. (For example, in FIG. 8, it can be configured to rotate around the irradiation position of incident light on the metal film 14 in a plane).

The sensor chip 30, the light source 52, and the light receiving means 58 constitute an SPR device for performing SPR measurement.

Above the sensor chip 30, there is provided a light detection means 60 for receiving the fluorescence 59 generated from the fluorescent substance that labels the analyte excited by the surface plasmon light (dense wave) generated on the metal film 14. ing.

The photodetection means 60 is not particularly limited. For example, an ultrasensitive photomultiplier tube, a CCD (Charge-Coupled Device) image sensor capable of multipoint measurement, and a CMOS (Complementary Metal-Oxide Semiconductor). An image sensor or the like can be used.

Note that a condensing member 62 for condensing light efficiently and a wavelength selection function member 64 formed so as to selectively transmit only the fluorescence 59 are provided between the sensor chip 30 and the light detection means 60. Is provided.

As the condensing member 62, any condensing system may be used as long as it aims at efficiently condensing the fluorescence on the light detecting means 60. As a simple condensing system, for example, a commercially available objective lens used in a microscope or the like may be used. The magnification of the objective lens is preferably 10 to 100 times.

As the wavelength selection function member 64, an optical filter, a cut filter, or the like can be used.
Examples of the optical filter include a neutral density (ND) filter and a diaphragm lens. Furthermore, as cut filters, external light (illumination light outside the device), excitation light (excitation light transmission component), stray light (excitation light scattering component in various places), plasmon scattered light (excitation light originated from, Scattered light generated by the influence of structures or deposits on the sensor chip surface), and various noise lights such as autofluorescence of the oxygen fluorescent substrate, such as interference filters and color filters. .

The sensor chip 30, the light source 52, and the light detection means 60 constitute an SPFS apparatus for performing SPFS measurement.

In the optical specimen detection apparatus 50 configured as described above, first, a ligand for capturing an analyte is solid-phased on the solid-phase film 16 of the sensor member 10. An appropriate amount of the antibody solution containing is fed and circulated for a predetermined time. As a result, the ligand for capturing the analyte is immobilized on the solid phase membrane.

Next, an appropriate amount of the sample liquid containing the analyte is fed and circulated for a predetermined time. As a result, the analyte is captured by the ligand solid-phased on the solid-phase film 16 on the metal film 14.

Next, an appropriate amount of a fluorescent substance solution containing a fluorescent substance for labeling the analyte is fed to the flow path 36 and circulated for a predetermined time. As a result, the analyte captured by the solid phase layer (solid phase film 16) on the metal film 14 is labeled with the fluorescent substance.

In this way, the analyte labeled with the fluorescent substance is fixed to the reaction area 10a of the sensor member 10. In this state, the excitation light 54 from the light source 52 is applied to the metal film 14 of the sensor member 10 via the dielectric member 32, and the metal film reflected light 55 is received by the light receiving means 58.

Then, by changing the incident angle of the excitation light 54 with respect to the metal film 14 by a predetermined angle, the relationship between the light intensity of the metal film reflected light 55 (hereinafter referred to as “SPR signal”) and the incident angle of the excitation light 54. SPR measurement can be performed by examining the above.

On the other hand, the excitation light 54 from the light source 52 is applied to the metal film 14 of the sensor member 10 via the dielectric member 32 and the light 59 is received by the light detection means 60, whereby the amount of fluorescence by SPFS measurement is measured. (Hereinafter referred to as “SPFS signal”).

By performing SPR measurement or SPFS measurement in this manner, for example, the total amount of analyte (analyte concentration) in the sample liquid is calculated by comparing with a calibration curve relating to the analyte concentration and SPFS signal created in advance. can do.

When the sensor member 10 of the present invention is used in such an optical specimen detection apparatus 50, as described above, after the sensor member 10 is attached to the dielectric member 32 to form the sensor chip 30, the optical member is detected. The sensor chip 30 may be configured by mounting the sensor member 10 on the dielectric member 32 mounted on the optical sample detection device 50.

The preferred embodiment of the present invention has been described above, but the present invention is not limited to this. For example, in the above embodiment, a sensor member is applied to an SPR device and an SPFS device as an optical specimen detection device. However, the present invention is not limited to this, and various modifications can be made without departing from the object of the present invention, such as application to other optical specimen detection devices.

FIG. 9 is a graph showing the relationship between the incident angle of the excitation light 54 measured using the sensor member 10 manufactured by the sensor member manufacturing method of the present invention and the SPR signal, and FIG. 10 shows the sensor member of the present invention. It is a graph which shows the relationship between the incident angle of the excitation light 54 measured using the sensor member 10 manufactured by this manufacturing method, and an SPFS signal.

In FIG. 9 and FIG. 11 described later, the ratio (reflectance) of the light amount of the excitation light 54 and the light amount of the metal film reflected light is used as the SPR signal. In FIG. 10 and FIG. 12 described later, the light quantity of the fluorescence 59 measured by the CCD sensor is used as the SPFS signal.

In this example, a ZEONOR (registered trademark) film (ZF14-188, refractive index: 1.53, thickness: 188 μm) manufactured by Nippon Zeon Co., Ltd. was used as the sensor substrate 12, and the metal film 14 was thickened by vapor deposition. It is formed as a thin film having a thickness of about 41 nm.

Further, as the dielectric member 32, a glass prism (BK7, refractive index: 1.5168) is used, and the sensor member 10 and the dielectric member 32 are made of a refractive index matching liquid (refractive index: 1.51). It is fixed through.

FIG. 11 is a comparative example, a graph showing the relationship between the incident angle of excitation light measured using a conventional sensor chip and the SPR signal, and FIG. 12 is a comparative example, using a conventional sensor chip. It is a graph which shows the relationship between the incident angle of the measured excitation light, and a SPFS signal.

In this comparative example, a metal film substrate provided with a metal film (thickness: about 42 nm) by vapor deposition on a glass substrate (BK7, thickness: 1000 μm, refractive index: 1.5168) is formed on a dielectric member. It is fixed.

Note that a glass prism (BK7, refractive index: 1.5168) is used as the dielectric member, and the metal film substrate and the dielectric member are connected via a refractive index matching liquid (refractive index: 1.51). It is fixed.

As shown in FIGS. 9 to 12, in the measurement using the sensor member manufactured by the sensor member manufacturing method of the present invention, both the SPR signal and the SPFS signal are almost the same as the measurement using the conventional glass substrate. The result was obtained.

For this reason, by using the manufacturing method of the sensor member of the present invention, it is possible to easily mass-produce while ensuring the same level of accuracy as before, and to reduce the production cost.

The present invention provides a sensor chip which is a member to be measured for an optical specimen detection device used in a field where high-precision measurement is required, such as clinical tests such as AFP sugar chain measurement and CEA sugar chain measurement. Cost and stability can be provided.

DESCRIPTION OF SYMBOLS 10 Sensor member 10a Reaction area 12 Sensor base material 14 Metal film 16 Solid phase film 18

Flexible base material

18a, 18b Both edge parts 19a, 19b Convex part 20 Flexible base material winding body 22 Base material feeding apparatus 24 group Material winding device 26 Metal film forming means 27 Solid phase film forming means 28 Base material cutting means 29 Adhesive 30 Sensor chip 32 Dielectric member

32b Side surface

32c Side surface 34 Reaction space forming member

34a Reaction space

35a Well member 35b Flow path member 36

Flow path

36a, 36b End 38 Refractive index matching liquid 40 Sensor chip holding member 42 Fastening member 44 Sealing member 50 Optical specimen detection device 52 Sensor chip loading part 52 Light source 54 Excitation light 55 Metal film reflected light 56 Polarization filter 58 Light receiving means 59 Fluorescence 60 Photodetection means 62 Condensing member 4 wavelength selecting function member 100 SPFS device 102 dielectric member 102a top 102b side 104 metal film 106 sensor chip 108 sensor chip loading section 110 light source 112 incident light 114 the channel 116 sensor area 118 fluorescence 120 light detecting means

Claims

A method for producing a sensor member for use in an optical sample detection apparatus that detects a sample by irradiating excitation light onto a metal film formed on a dielectric member,
Forming a metal film on a flexible substrate having translucency;
Forming a solid phase film used for immobilizing the specimen on the metal film formed on the flexible substrate;
Cutting the flexible base material on which the metal film and the solid phase film are formed into a predetermined size;
The manufacturing method of the sensor member containing this.
The metal film is sequentially applied onto the flexible substrate by sequentially feeding out the flexible substrate from a flexible substrate wound body on which the long flexible substrate is wound. The solid phase film is sequentially formed on the metal film, and the flexible base material on which the metal film and the solid phase film are formed is sequentially cut into a predetermined size. Manufacturing method of the sensor member.
The method for producing a sensor member according to claim 2, wherein convex portions are provided on both edges in the width direction of the flexible base material.
The method for producing a sensor member according to any one of claims 1 to 3, wherein the metal film is formed by a sputtering method or a vapor deposition method.
The method for producing a sensor member according to any one of claims 1 to 4, wherein the solid phase film is formed by inkjet coating.
The method for producing a sensor member according to any one of claims 1 to 5, wherein the flexible base material on which the metal film and the solid phase film are formed is cut into a predetermined size by punching.
The method for producing a sensor member according to claim 6, wherein the flexible base material on which the metal film and the solid phase film are formed is punched into a circular shape.
The method for manufacturing a sensor member according to claim 6 or 7, wherein a punching process is performed along the optical axis of the flexible substrate.
The method for producing a sensor member according to claim 8, wherein the flexible substrate is a polyethylene terephthalate film.
A method of manufacturing a sensor chip for use in an optical sample detection device that detects a sample by irradiating excitation light onto a metal film formed on a dielectric member,
Fixing the sensor member manufactured by the method for manufacturing a sensor member according to any one of claims 1 to 9 on the dielectric member;
A sample solution containing the sample is injected onto the dielectric member to which the sensor member is fixed, and a reaction space forming member for forming a reaction space for causing the reaction between the solid phase film and the sample is fixed. Process,
A method of manufacturing a sensor chip including:
The method for manufacturing a sensor chip according to claim 10, wherein a refractive index matching liquid is interposed between the dielectric member and the sensor member.
The method for manufacturing a sensor chip according to claim 11, wherein the refractive index matching liquid is an adhesive.
The method for manufacturing a sensor chip according to any one of claims 10 to 13, wherein the dielectric member and the reaction space forming member are fixed by a fastening member.
The dielectric member and the sensor member are sandwiched between the sensor chip holding member and the reaction space forming member in a state where the dielectric member is placed on the sensor chip holding member, and fixed by a fastening member. Item 14. A method for producing a sensor chip according to any one of Items 10 to 13.
The sensor according to any one of claims 10 to 14, wherein a seal member is placed so as to surround the sensor member, and is fixed so as to sandwich the seal member by the reaction space forming member and the dielectric member. Chip manufacturing method.
The method for manufacturing a sensor chip according to any one of claims 10 to 15, wherein the reaction space forming member is a well member capable of temporarily storing the sample liquid.
The method for manufacturing a sensor chip according to any one of claims 10 to 15, wherein the reaction space forming member is a flow path member capable of circulating the sample liquid with respect to a reaction area of the sensor member.
The method for manufacturing a sensor chip according to any one of claims 10 to 17, wherein the dielectric member is formed of a natural polymer or a synthetic polymer.
A method of using a sensor member used in an optical specimen detection device having a dielectric member and a light source disposed on one side surface below the dielectric member,
Attaching the sensor member produced by the method for producing a sensor member according to any one of claims 1 to 9 on the dielectric member;
Irradiating the metal film of the sensor member with excitation light from the light source;
Method of using a sensor member having
The method for using a sensor member according to claim 18, wherein the optical specimen detection device further includes a light detection means disposed above the dielectric member.
The method of using a sensor member according to claim 18, wherein the optical specimen detection device further includes a light receiving means disposed on the other side surface below the dielectric member.