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WO2014178385A1 - Dispositif de capture de substance cible et dispositif de détection de substance cible - Google Patents

Dispositif de capture de substance cible et dispositif de détection de substance cible Download PDF

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Publication number
WO2014178385A1
WO2014178385A1 PCT/JP2014/061899 JP2014061899W WO2014178385A1 WO 2014178385 A1 WO2014178385 A1 WO 2014178385A1 JP 2014061899 W JP2014061899 W JP 2014061899W WO 2014178385 A1 WO2014178385 A1 WO 2014178385A1
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WO
WIPO (PCT)
Prior art keywords
target substance
photonic crystal
metal film
light
support member
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2014/061899
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English (en)
Japanese (ja)
Inventor
横山　景介
古川　秀樹
暢子 奥谷
邦彦 笹尾
寿明 小口
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
NSK Ltd
Original Assignee
NSK Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP2013095947A external-priority patent/JP2014215289A/ja
Priority claimed from JP2013095974A external-priority patent/JP2014215291A/ja
Priority claimed from JP2014025826A external-priority patent/JP2014232098A/ja
Application filed by NSK Ltd filed Critical NSK Ltd
Priority to US14/784,604 priority Critical patent/US20160069798A1/en
Publication of WO2014178385A1 publication Critical patent/WO2014178385A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material

Definitions

  • the present invention relates to a target substance capturing device that detects a target substance and a target substance detection device including the target substance capturing device.
  • Biosensors using photonic crystals are known as means for detecting target substances such as proteins and cells and measuring concentrations (for example, Non-Patent Document 1 and Non-Patent Document 2).
  • the biosensor described in Non-Patent Document 1 and Non-Patent Document 2 irradiates light on a photonic crystal substrate on which a gold thin film is formed, and changes the wavelength peak of reflected light reflected by the photonic crystal substrate. By measuring, the target substance is detected or the concentration of the target substance is measured.
  • the attachment state When detecting a target substance using a biosensor, if the biosensor is removed and then attached again by changing the liquid to be detected, the attachment state may be different. If the attachment state of the biosensor is different, the detection sensitivity of the target substance may decrease due to this.
  • the photonic crystal has a fine structure, it is difficult to precisely control the shape even in the same manufacturing process. For this reason, variation exists for each sensor, and there is a possibility that the measurement accuracy of the target substance is lowered.
  • Non-Patent Document 2 describes that real-time measurement using a biosensor was performed.
  • the reflected light of the light irradiated to the photonic crystal substrate exposed to the solution in the flow path is observed at regular intervals.
  • the change in the reflected light of the light applied to the photonic crystal substrate becomes faster as the flow rate of the solution increases.
  • the flow rate of the solution is increased, the amount of the solution that passes through without reacting with the photonic crystal substrate increases, and the amount of the solution required to reach the equilibrium state increases. Therefore, there is a demand for a target substance capturing device that can reduce the amount of solution required to reach an equilibrium state while rapidly changing the reflected light of the light irradiated to the photonic crystal substrate.
  • the present invention can reduce the amount of solution required to reach an equilibrium state while suppressing a decrease in detection sensitivity of the target substance and speeding up the change in reflected light of the light irradiated to the photonic crystal substrate.
  • An object is to provide at least one of providing a target substance capturing device.
  • the present invention provides a support member having a metal film covering structure that captures a target substance placed thereon and supported, and a support member having at least two holes opened in a portion different from the portion on which the metal film covering structure is placed.
  • the metal film covering structure is sandwiched between the support member and the hole of the support member and the portion of the metal film covering structure placed on the support member that captures the target substance overlap.
  • the target substance capturing device includes a holding member having a portion and a covering member that has translucency and covers the opening of the holding member. In this way, even if the liquid to be detected is changed, it is not necessary to remove the metal film covering structure, so that the detection sensitivity of the target substance is reduced due to different mounting states of the metal film covering structure. Can be suppressed.
  • the hole includes two holes, a supply hole for supplying a liquid containing the target substance to a space surrounded by the covering member, the inner surface of the opening, and the support member, and a discharge hole for discharging the liquid from the space. It is preferable that If it does in this way, a liquid can be supplied to an opening part and a liquid can be discharged
  • the portion of the holding member that contacts the metal film covering structure is preferably made of at least silicone, and more preferably made of polydimethylsiloxane. In this way, the metal film covering structure can be easily removed from the holding member.
  • the support member is preferably made of a fluororesin. In this way, the metal film covering structure can be easily detached from the support member.
  • the support member preferably has translucency. In this way, not only the reflected light of the light irradiated to the metal film covering structure but also the transmitted light can be observed.
  • the support member preferably has a plurality of claws engaged with the holding member sandwiching the metal film covering structure between the support member and the support member on the side on which the metal film covering structure is placed. In this way, the holding member and the covering member can be easily attached to the support member, and the holding member and the covering member can be easily detached from the support member.
  • the covering member is fitted into the opening of the holding member. In this way, the holding member and the covering member can be easily attached to the support member, and the holding member and the covering member can be easily detached from the support member.
  • the present invention detects the reflected light of the parallel light reflected by the target substance capturing device and the part that captures the target substance from the opening and reflected by the part that captures the target substance.
  • a liquid feeding device that supplies the liquid to the space through the hole and discharges the liquid from the space through the hole. If it does in this way, a liquid can be easily supplied to the opening part of a holding member, and a liquid can be easily discharged
  • the light detection unit includes a first spectrometer and a second spectrometer having a higher wavelength resolution of light that can be detected than the first spectrometer, and the processing unit uses the first spectrometer. After obtaining the wavelength of the extreme value of the reflected light, the wavelength of the extreme value of the reflected light is obtained within the range of the wavelength of the extreme value obtained by the first spectrometer using the second spectrometer. preferable. In this way, the extreme wavelength of the reflected light can be obtained quickly and accurately.
  • the extreme wavelength of the reflected light by fitting at least one of the detection result of the first spectrometer and the detection result of the second spectrometer as a function. In this way, since the resolution higher than the pixel resolution of the spectroscope can be realized, the wavelength of the reflected light at the extreme value can be obtained more accurately.
  • the present invention provides a support member for placing and supporting a metal film coating structure for capturing a target substance, and sandwiching the metal film coating structure between the support member, and the metal film coating structure
  • a holding member having a plurality of openings overlapping a portion that captures a target substance, a translucent covering member that covers the opening of the holding member, and the support member, the metal film covering structure
  • the target substance capturing device includes a hole that is open to each of the two openings with respect to one of the openings in a state where a body is sandwiched between the holding member and the support member. Since this target substance capturing device can introduce liquid into each opening of the support member, the metal film covering structure can be calibrated simultaneously with the inspection. As a result, this target substance capturing device can realize highly accurate measurement.
  • one opening is provided with a supply hole for supplying a liquid containing the target substance to the opening and a discharge hole for discharging the liquid from the opening. If it does in this way, a liquid can be supplied to each opening part and a liquid can be discharged
  • the portion of the holding member that contacts the metal film covering structure is preferably made of at least silicone, and more preferably made of polydimethylsiloxane. In this way, the metal film covering structure can be easily removed from the holding member.
  • the support member is preferably made of a fluororesin. In this way, the metal film covering structure can be easily detached from the support member.
  • the present invention is provided for the target substance capturing device described above and each of the openings, and irradiates parallel light to the part that captures the target substance from each of the openings, thereby capturing the target substance.
  • a light detection unit that detects reflected light of the parallel light reflected by the part, and obtains the wavelength of the extreme value of the reflected light detected by the light detection unit, and based on the obtained shift of the wavelength of the extreme value
  • a target substance detection device including at least a processing unit that detects the presence or absence of the target substance. Since this target substance detection device includes the target substance capturing device described above, it is possible to suppress a decrease in the detection accuracy of the target substance.
  • a liquid feeding device that supplies the liquid to the space through the hole and discharges the liquid from the space through the hole. If it does in this way, a liquid can be easily supplied to each opening part which a holding member has, and a liquid can be easily discharged
  • the present invention includes a flow path through which a fluid containing a target substance flows, and a substrate having a reflective surface that captures the target substance and reflects irradiated light, and the substrate has at least a part of the fluid.
  • the target substance capturing device wherein the fluid is disposed in the flow path so as to pass through the reflection surface, and the fluid that has passed through the flow path is repeatedly guided to the flow path.
  • the target substance capturing device repeatedly introduces fluid to the reflecting surface.
  • the solution which passed without reacting with a photonic crystal substrate can be repeatedly obtained the opportunity to react with a photonic crystal substrate.
  • increasing the flow rate of the solution does not increase the amount of solution required to reach equilibrium. Therefore, the target substance detection apparatus according to the present invention can reduce the amount of fluid required to reach the equilibrium state while speeding up the change in the reflected light of the light irradiated to the photonic crystal substrate.
  • the flow path includes a supply port through which the fluid flows and a discharge port through which the fluid flows out, and the fluid discharged from the discharge port may be guided to the flow path from the supply port.
  • the motive power for making a fluid flow can be installed in the exterior of a flow path. Since the channel is very small, if the power can be installed outside the channel, the target substance capturing device can be easily assembled. Therefore, the target substance capturing device according to the present invention is easy to assemble, and further reduces the amount of solution required to reach an equilibrium state, while making the change in the reflected light of the light irradiated to the photonic crystal substrate faster. Less.
  • the apparatus further includes a container for storing a new fluid containing the target substance, and the new fluid is guided from the supply port to the flow path.
  • a plate-shaped table a thin plate having an opening portion, which overlaps the plate in a direction perpendicular to the surface of the table, and a plate on the plate in a direction perpendicular to the surface of the table.
  • the flow path is a space surrounded by the base, the inner wall of the opening, and the cover.
  • the supply port and the discharge port are preferably through holes provided in the table.
  • the present invention can reduce the amount of solution required to reach an equilibrium state while suppressing a decrease in detection sensitivity of the target substance and speeding up the change in reflected light of the light irradiated to the photonic crystal substrate. It is possible to realize at least one of providing a target substance capturing device.
  • FIG. 1 is a diagram illustrating a target substance detection device including the target substance capturing device according to the first embodiment.
  • FIG. 2 is a side view of the photonic crystal biosensor according to the first embodiment.
  • FIG. 3 is a perspective view of the photonic crystal biosensor according to the first embodiment.
  • FIG. 4 is a plan view of the photonic crystal biosensor according to the first embodiment.
  • FIG. 5 is a perspective view of a metal film-coated photonic crystal.
  • FIG. 6 is a plan view of a metal film-coated photonic crystal.
  • FIG. 7 is a diagram showing a cross section when the photonic crystal is cut along a plane orthogonal to the surface of the photonic crystal.
  • FIG. 8 is a diagram showing an AA cross section in FIG.
  • FIG. 10 is a diagram illustrating a method for manufacturing a photonic crystal.
  • FIG. 11 is a diagram illustrating a method for manufacturing a photonic crystal.
  • FIG. 12 is a diagram illustrating a method for manufacturing a photonic crystal.
  • FIG. 13 is a diagram for explaining the principle of the photonic crystal biosensor.
  • FIG. 14 is a diagram for explaining the principle of a photonic crystal biosensor.
  • FIG. 15 is a diagram for explaining the principle of the photonic crystal biosensor.
  • FIG. 16 is a diagram for explaining the principle of the photonic crystal biosensor.
  • FIG. 10 is a diagram illustrating a method for manufacturing a photonic crystal.
  • FIG. 11 is a diagram illustrating a method for manufacturing a photonic crystal.
  • FIG. 12 is a diagram illustrating a method for manufacturing a photonic crystal.
  • FIG. 13 is a diagram for explaining the principle of the photonic crystal biosensor.
  • FIG. 14 is a diagram for explaining the principle of a photonic
  • FIG. 17 is a diagram illustrating the relationship between the intensity of the extreme value of reflected light and the wavelength.
  • FIG. 18 is a diagram showing the relationship between the wavelength shift amount at the extreme value of the intensity of the reflected light and the concentration of avidin immobilized on the reflecting surface of the photonic crystal using biotin.
  • FIG. 19 is a diagram illustrating a structure of a measurement probe included in the light detection unit illustrated in FIG.
  • FIG. 20 is a diagram illustrating a spectroscope pixel included in the photodetection device.
  • FIG. 21 is a diagram illustrating a spectroscope pixel included in the photodetection device.
  • FIG. 22 is a diagram illustrating an example of a spectrum of reflected light detected by the spectroscope illustrated in FIG. FIG.
  • FIG. 23 is a diagram illustrating an example of a spectrum of reflected light detected by the spectroscope illustrated in FIG.
  • FIG. 24-1 is a diagram illustrating an example of a spectrum of reflected light when the light detection element included in the spectroscope is not cooled.
  • FIG. 24-2 is a diagram illustrating an example of a spectrum of reflected light when the light detection element included in the spectroscope is cooled.
  • FIG. 25A is a diagram illustrating an example of a spectrum of reflected light detected by a light detection element included in the spectroscope.
  • FIG. 25-2 is a diagram for explaining an example of obtaining the peak position by data fitting the result shown in FIG. 25-1.
  • FIG. 25-1 is a diagram illustrating an example of a spectrum of reflected light detected by the spectroscope illustrated in FIG.
  • FIG. 25-1 is a diagram illustrating an example of a spectrum of reflected light when the light detection element included in the spectroscope is not cooled.
  • FIG. 25C is a diagram illustrating a peak position obtained from the detection result of the light detection device and a peak position obtained by fitting detection result data of the light detection device.
  • FIG. 25-4 is a diagram illustrating a peak position obtained from the detection result of the light detection device and a peak position obtained by fitting detection result data of the light detection device.
  • FIG. 25-5 is a diagram illustrating a change with time of the peak wavelength obtained from the detection result of the light detection device.
  • FIG. 25-6 is a diagram showing a change with time of the peak wavelength obtained by peak fitting the detection result of the photodetection device.
  • FIG. 25-7 is a flowchart showing each peak fitting process.
  • FIG. 26 is a diagram illustrating a modified example of the liquid handling unit.
  • FIG. 26 is a diagram illustrating a modified example of the liquid handling unit.
  • FIG. 27 is a diagram illustrating a modified example of the liquid handling unit.
  • FIG. 28 is a diagram illustrating a first modification of the photonic crystal biosensor.
  • FIG. 29 is a diagram illustrating a first modification of the photonic crystal biosensor.
  • FIG. 30 is a diagram illustrating a first modification of the photonic crystal biosensor.
  • FIG. 31 is a diagram illustrating a second modification of the photonic crystal biosensor.
  • FIG. 32 is a diagram illustrating a second modification of the photonic crystal biosensor.
  • FIG. 33 is a diagram illustrating a photonic crystal biosensor according to the second embodiment.
  • FIG. 34 is a diagram illustrating a photonic crystal biosensor according to the second embodiment.
  • FIG. 35 is a perspective view showing a light detection unit according to the second embodiment.
  • FIG. 36 is an exploded view of the light detection unit according to the second embodiment.
  • FIG. 37 is an exploded view of the light detection unit according to the second embodiment.
  • FIG. 38 is a diagram showing a target substance detection device.
  • FIG. 39 is an explanatory diagram of a photonic crystal biosensor.
  • FIG. 40 is a diagram illustrating a state before the solution is supplied to the flow path.
  • FIG. 41 is a diagram showing a state in which a solution is circulated.
  • FIG. 42 is a flowchart showing an example of a solution circulation method.
  • FIG. 43 is an explanatory diagram of another circulation method.
  • FIG. 44 is a diagram illustrating a change in the wavelength of the extreme value of reflected light with respect to time in the example and the comparative example.
  • FIG. 44 is a diagram illustrating a change in the wavelength of the extreme value of reflected light with respect to time in the example and the comparative example.
  • FIG. 45 is a diagram illustrating the evaluation conditions of the light detection unit of the target substance detection device.
  • FIG. 46 is a flowchart of the target substance detection method.
  • FIG. 47 is a diagram for explaining the principle of the photonic crystal biosensor.
  • FIG. 48 is a diagram for explaining the principle of the photonic crystal biosensor.
  • FIG. 49 is a diagram for explaining the principle of the photonic crystal biosensor.
  • FIG. 50 is a diagram for explaining the principle of the photonic crystal biosensor.
  • FIG. 51 is a diagram for explaining the principle of the photonic crystal biosensor.
  • FIG. 1 is a diagram illustrating a target substance detection device including the target substance capturing device according to the first embodiment.
  • the target substance detection device 10 includes a photonic crystal biosensor 11 as a target substance capturing device, a light detection unit 12, a processing unit 13, and a liquid handling unit 14. First, the photonic crystal biosensor 11 will be described.
  • FIG. 2 is a side view of the photonic crystal biosensor according to the first embodiment.
  • FIG. 3 is a perspective view of the photonic crystal biosensor according to the first embodiment.
  • FIG. 4 is a plan view of the photonic crystal biosensor according to the first embodiment.
  • the photonic crystal biosensor 11 includes a holding device 11H and a metal film-covered photonic crystal 21 as a metal film-covering structure.
  • the holding device 11H holds the metal film-coated photonic crystal 21.
  • the holding device 11 ⁇ / b> H includes a covering member 22, a holding member 23, and a support member 24.
  • the holding device 11H holds the metal film-coated photonic crystal 21.
  • the holding device 11 ⁇ / b> H holds the metal film-coated photonic crystal 21 placed on the support member 24 while the holding member 23 is sandwiched between the support member 24.
  • the covering member 22 covers the surface of the holding member 23 on the side opposite to the support member 24.
  • the support member 24, the holding member 23, and the covering member 22 are plate-like members.
  • the shape of the support member 24, the holding member 23, and the covering member 22 is a rectangle (including a square) when viewed from a direction orthogonal to the surfaces, that is, in plan view.
  • the shapes of the support member 24, the holding member 23, and the covering member 22 are not limited to a rectangle, and may be a polygon such as a hexagon, a circle, or the like. By making the shape of the support member 24, the holding member 23, and the covering member 22 rectangular, there are advantages such as easy manufacture and easy attachment of the holding device 11H to the attachment jigs 27 and 28 shown in FIG.
  • the support member 24 mounts and supports the metal film-coated photonic crystal 21. As shown in FIGS. 1 to 4, the support member 24 has at least two holes 24 ⁇ / b> HI and 24 ⁇ / b> HE that open to a portion different from the portion on which the metal film-covered photonic crystal 21 is placed.
  • the holding member 23 sandwiches the metal film-covered photonic crystal 21 between the support member 24 and the support member 24.
  • the holding member 23 has an opening 23P. As shown in FIG. 2, the opening 23 ⁇ / b> P passes through the two opposing flat surfaces 23 ⁇ / b> UP and 23 ⁇ / b> DP that are the largest of the holding member 23 that is a plate-like member. As shown in FIGS.
  • the opening 23 ⁇ / b> P is a channel having a rectangular shape in plan view and a groove shape. As shown in FIG. 4, the opening 23 ⁇ / b> P overlaps the holes 24 ⁇ / b> HI and 24 ⁇ / b> HE of the support member 24 and a portion 21 ⁇ / b> C that captures the target substance of the metal film-coated photonic crystal 21 placed on the support member 24.
  • the hole 24HI supplies a liquid such as a solution containing a target substance capturing substance into the opening 23P.
  • the hole 24HE discharges a liquid such as a solution containing the target substance capturing substance from the opening 23P.
  • the holes 24HI are appropriately referred to as supply holes 24HI, and the holes 24HE are referred to as discharge holes 24HE.
  • the covering member 22 covers the opening 23P of the holding member 23 as shown in FIGS.
  • the covering member 22 has translucency. This is because the metal film-covered photonic crystal 21 is irradiated with light through the covering member 22, and the change in the wavelength peak of the reflected light reflected by the metal film-covered photonic crystal 21 is measured. This is because the substance is detected or the concentration of the target substance is measured.
  • the covering member 22 is, for example, a glass plate, a transparent resin plate, or a transparent resin film.
  • a liquid such as a solution containing a target substance capturing substance is supplied from the supply hole 24HI and held in the space 23SP.
  • the liquid held in the space 23SP contacts the portion 21C that captures the target substance of the metal film-coated photonic crystal 21.
  • the liquid held in the space 23SP is held in the space 23SP while the target substance detection device 10 detects the target substance or measures the concentration of the target substance. After the target substance detection apparatus 10 detects the target substance, the liquid held in the space 23SP is discharged from the discharge hole 24HE.
  • a liquid supply pipe 25 is connected to the photonic crystal biosensor 11 in order to supply liquid from outside the photonic crystal biosensor 11 into the space 23SP.
  • a liquid discharge pipe 26 is connected to the photonic crystal biosensor 11.
  • the support member 24 having the supply hole 24HI has a hole 24Hsi and a hole 24Hse on the surface opposite to the surface on which the metal film-covered photonic crystal 21 is placed.
  • the hole 24Hsi is connected to the supply hole 24HI.
  • 24Hse is connected to the discharge hole 24HE.
  • the hole 24Hsi, the hole 24Hse, the supply hole 24HI, and the supply hole 24HE have a circular cross section.
  • the diameter of the hole 24Hsi is larger than the diameter of the supply hole 24HI.
  • the diameter of the hole 24Hse is larger than the diameter of the discharge hole 24HE.
  • a connecting member 25S that connects the supply hole 24HI and the liquid supply pipe 25 is attached to the hole 24Hsi.
  • a connecting member 26S that connects the discharge hole 24HE and the liquid discharge pipe 26 is attached to the hole 24Hse.
  • the connection members 25S and 26S are, for example, rubber, resin, or metal.
  • the connection members 25S and 26S have attachment holes 25SH and 26SH, respectively.
  • the liquid supply pipe 25 is inserted into the attachment hole 25SH and attached to the connection member 25S.
  • the liquid discharge pipe 26 is inserted into the attachment hole 26SH and attached to the connection member 26S.
  • the liquid supply pipe 25 is attached to the hole 24Hsi of the support member 24 via the connection member 25S. Further, the liquid discharge pipe 26 is attached to the hole 24Hse of the support member 24 through the connection member 26S.
  • the hole 24Hsi to which the liquid supply pipe 25 is attached is connected to the supply hole 24HI, and the hole 24Hse to which the liquid discharge pipe 26 is attached is connected to the discharge hole 24HE.
  • the liquid supply pipe 25 is connected to the space 23SP through the connection member 25S and the supply hole 24HI.
  • the liquid discharge pipe 26 is connected to the space 23SP through the connection member 26S and the discharge hole 24HE.
  • the photonic crystal biosensor 11 holds the metal film-covered photonic crystal 21 between the support member 24 and the holding member 23.
  • the attachment jigs 27 and 28 are fastened by a bolt 29 shown in FIG. 3 in a state where the support member 24 and the holding member 23 sandwiching the metal film-coated photonic crystal 21 are sandwiched.
  • the photonic crystal biosensor 11 is sandwiched and supported by the mounting jigs 27 and 28 shown in FIG. 3 with the covering member 22 attached to the holding member 23. Since the support member 24, the holding member 23, and the metal film-covered photonic crystal 21 can be integrated with the mounting jigs 27 and 28, handling becomes easy. Further, by fastening the mounting jigs 27 and 28 with the bolt 29, the photonic crystal biosensor 11 can be easily disassembled.
  • the fixing of the mounting jigs 27 and 28 is not limited to the fastening by the bolt 29.
  • the holding member 23 is formed of at least a silicone, for example, polydimethylsiloxane (PDMS), at a portion in contact with the metal film-coated photonic crystal 21. Since polydimethylsiloxane has high liquid repellency (water repellency), adsorption between the metal film-coated photonic crystal 21 and the holding member 23 can be suppressed. For this reason, the metal film-coated photonic crystal 21 can be easily detached from the holding member 23 when the metal film-coated photonic crystal 21 is replaced.
  • the thickness of the holding member 23 is preferably 100 ⁇ m or more and 2 mm or less. This facilitates handling when the metal film-coated photonic crystal 21 is fixed between the support member 24 and the holding member 23.
  • the thickness of the interval holding portion depends on the wavelength of plasmon resonance, the thickness is limited by the wavelength band to be used. With such a structure, the thickness of the interval holding portion needs to be strictly accurate, and time and manufacturing cost are required at the time of manufacturing. In the present embodiment, since the interval holding unit is unnecessary, the photonic crystal biosensor 11 can be easily manufactured, and the time and manufacturing cost can be suppressed.
  • the surface plasmon (SPR) sensor it is important to fix the sensor and the prism, and there is a problem that when a slight gap or bending occurs, the sensor does not function as a sensor.
  • the crystal 21 does not need to be fixed as strictly as the SPR sensor.
  • the support member 24 is made of a fluororesin.
  • the material of the support member 24 is not limited to the fluororesin, but since the fluororesin has high liquid repellency (water repellency), adsorption between the metal film-covered photonic crystal 21 and the support member 24 can be suppressed. For this reason, the metal film-coated photonic crystal 21 can be easily detached from the holding member 23 when the metal film-coated photonic crystal 21 is replaced.
  • the support member 24 may have translucency. By doing in this way, the transmitted light of the light irradiated to the metal film covering photonic crystal 21 can also be observed.
  • the support member 24 has translucency, the support member 24 is manufactured using glass or transparent resin, for example.
  • the metal film-covered photonic crystal 21 is fixed between the support member 24 and the holding member 23 by a joining technique such as thermal fusion. Next, the metal film-coated photonic crystal 21 will be described.
  • FIG. 5 is a perspective view of a metal film-coated photonic crystal.
  • FIG. 6 is a plan view of a metal film-coated photonic crystal.
  • FIG. 7 is a diagram showing a cross section taken along the line AA in FIG.
  • FIG. 7 shows a cross section when the photonic crystal is cut along a plane orthogonal to the surface of the photonic crystal.
  • FIG. 9 described later. 5 to 9 are schematic diagrams, and therefore the thickness and size of each element of the metal film-covered photonic crystal 21 are different from actual ones. The same applies hereinafter.
  • the metal film-coated photonic crystal 21 captures the target substance. As shown in FIGS.
  • the metal film-covered photonic crystal 21 includes a photonic crystal 65 and a metal film 66.
  • the metal film 66 covers a reflection surface 69 in which concave portions (hereinafter simply referred to as concave portions) 68 ⁇ / b> A having a circular cross section are formed on the surface 67 of the photonic crystal 65.
  • a photonic crystal has a reflective surface in which concave portions with a predetermined depth or convex portions with a predetermined height are periodically formed on the surface, and when the reflective surface is irradiated with light of a specific wavelength (parallel light), the reflection A structure from which light is obtained.
  • a structure that obtains reflected light of a specific wavelength when light is irradiated onto a reflective surface having concave portions or convex portions formed periodically on the surface is generally called a photonic crystal.
  • a photonic crystal is a structure having a lattice structure with sub-wavelength intervals. And when it irradiates the surface of a structure (henceforth a reflective surface) with the light of a wide region wavelength, it reflects or permeate
  • the surface state of the photonic crystal depends on, for example, the shape and material of the photonic crystal. By reading the change in the reflected light or transmitted light, the change in the surface state of the photonic crystal can be quantified. Examples of changes in the surface state of the photonic crystal include adsorption of substances on the surface, structural changes, and the like.
  • an extreme value appears in the light reflectance or light transmittance.
  • This extreme value of reflectance or transmittance depends on the type of metal, the thickness of the metal, and the surface shape of the photonic crystal.
  • the change in the surface state of the photonic crystal can be quantified by reading the light reflectance or light transmittance.
  • the metal thin film will be described later. In order to quantify the change in the surface state of the photonic crystal from the change in reflected light or transmitted light, the following method can be used.
  • the amount of change in reflectance or transmittance at an extreme value (maximum value or minimum value) or the shift amount of a wavelength at which the reflectance or transmittance becomes an extreme value is obtained.
  • the change in the surface state of the photonic crystal can be quantified by obtaining the amount of change with respect to the extreme value of interest or by obtaining the amount of shift of the wavelength that is the extreme value of interest.
  • the photonic crystal 65 has a reflecting surface 69 in which concave portions (non-flat portions) 68A are periodically formed on the surface 67.
  • the reflecting surface 69 is irradiated with light, light having a specific wavelength depending on the shape and material of the photonic crystal 65 is reflected.
  • the recesses 68A are arranged in a triangular lattice shape in plan view.
  • the diameter D1 of the recessed part 68A is 50 to 1000 nm, More preferably, it is 100 to 500 nm.
  • the distance C1 between the centers of the recesses 68A is preferably 100 nm or more and 2000 nm or less, and more preferably 200 nm or more and 1000 nm or less.
  • the aspect ratio (H1 / D1) of the recess 68A is preferably 0.1 or more and 10 or less, more preferably 0.5 or more and 5.0 or less. is there.
  • the dimension of the recess 68A is not limited to the above.
  • the shape and dimensions of the photonic crystal 65 are not limited to the shapes shown in FIGS. For example, a rectangular or polygonal lattice pattern formed on the surface, or a parallel line pattern or corrugated pattern formed on the surface (specifically, a pattern or the like formed periodically) ) Or a combination of these patterns.
  • a material of the photonic crystal 65 an organic material such as a synthetic resin or an inorganic material such as a metal or ceramic can be used.
  • Synthetic resins include polyethylene, polypropylene, polymethylpentene, polycycloolefin, polyamide, polyimide, acrylic, polymethacrylic acid ester, polycarbonate, polyacetal, polytetrafluoroethylene, polybutylene terephthalate, polyethylene terephthalate, polyvinyl chloride, polychlorinated Thermosetting resins such as vinylidene, polystyrene, polyphenylene sulfide, polyethersulfone, polyetheretherketone, and the like, and phenol resins, urea resins, and epoxy resins can be used.
  • ceramics such as silica, alumina, zirconia, titania and yttria can be suitably used.
  • metal various alloys including steel materials can be used. Specifically, stainless steel, titanium, a titanium alloy, or the like can be preferably used.
  • polycycloolefin-based synthetic resins or silica-based ceramics are more preferable.
  • the polycycloolefin synthetic resin is most suitable because of its excellent processability.
  • the photonic crystal 65 is manufactured by performing fine processing on the surface of the material substrate described above.
  • a processing method laser processing, thermal nanoimprint, optical nanoimprint, a combination of a photomask and etching, or the like can be used.
  • a thermoplastic resin such as a polycycloolefin-based synthetic resin
  • a method using thermal nanoimprinting is preferable.
  • the metal film 66 will be described.
  • the reflective surface 69 of the photonic crystal 65 is covered with a metal film 66.
  • the metal film 66 is preferably formed using one or more of gold (Au), silver (Ag), platinum (Pt), and aluminum (Al).
  • the metal film 66 is made of Au.
  • Au is preferable as the reflecting surface 69 because it is excellent in stability.
  • the surface is preferably covered with gold. By doing in this way, the usage-amount of gold
  • the thickness of the metal film 66 is small, part of the incident light on the photonic crystal 65 may pass through the metal film 66. As a result, there is a possibility that a lot of unnecessary information is included in the reflected light from the photonic crystal 65, such as a decrease in the amount of information obtained from the reflected light, diffracted light, or reflected light from the back surface of the photonic crystal 65.
  • unnecessary information contained in the reflected light from the photonic crystal 65 can be reduced, and the detection accuracy of the target substance and the concentration measurement accuracy can be improved. Further, it is preferable that the thickness of the metal film 66 is moderately small because a detailed pattern shape can be easily formed on the surface 67 of the photonic crystal 65.
  • the thickness of the metal film 66 is preferably 30 nm or more and 1000 nm or less, more preferably 150 nm or more and 500 nm or less, and further preferably 200 nm or more and 400 nm or less. This is because the change of the reflectance with respect to the wavelength becomes almost the same when the thickness of the metal film 66 exceeds 200 nm.
  • the metal film 66 can be formed on the reflection surface 69 of the photonic crystal 65 by sputtering or vapor deposition.
  • the outermost surface of the metal film 66 is preferably Au.
  • Ag, Pt, or Al is used for the metal film 66, the wavelength of the reflected light at each extreme value is 1.5 times that when Au is used as the metal film 66.
  • Ag, Pt, and Al have a sensitivity that is 1.5 times that of Au. Since Ag is easily oxidized, it is preferable to form an oxide thin film such as Au or SiO 2 that is not easily oxidized after forming Ag on the reflection surface 69 of the photonic crystal 65.
  • an Au film having a thickness of 5 nm can be formed on the surface of the Ag film having a thickness of 200 nm.
  • the sensitivity is 1.5 times that of an Au film having a thickness of 200 nm. Further, no change in sensitivity was observed with or without the 5 nm Au film.
  • Al is also easily oxidized like Ag, after forming an Al film on the surface 67 of the photonic crystal 65, it is preferable to form an oxide thin film such as Au or SiO 2 that is not easily oxidized.
  • Pt also preferably forms an oxide thin film such as Au or SiO 2 .
  • the reflective surface 69 of the photonic crystal 65 is preferably modified using 3-triethoxysilylpropylamine (APTES) or the like.
  • APTES 3-triethoxysilylpropylamine
  • the Au or Ag metal film 66 is formed on the reflective surface 69 of the photonic crystal 65, it is not APTES but has a thiol group at one end and a functional group such as an amino group or a carboxyl group at the other end. It is preferable to modify the reflection surface 69 of the photonic crystal 65 by using the carbon chain that it has.
  • a silane coupling agent having a functional group at one end for example, APTES is used to reflect the reflective surface of the photonic crystal 65. 69 is preferably modified.
  • the metal film-coated photonic crystal 21 is obtained by coating the reflective surface 69 of the photonic crystal 65 with the metal film 66, the metal film-coated photonic crystal is formed on the reflective surface 69 corresponding to the recess 68A of the photonic crystal 65.
  • 21 concave portions (non-flat portions) 68B are periodically formed.
  • the recesses 68B are arranged in a triangular lattice pattern, similar to the recesses 68A.
  • the diameter D2 of the recessed part 68B is based also on the thickness of the metal film 66, it is preferable that they are 50 nm or more and 1000 nm or less, More preferably, they are 100 nm or more and 500 nm or less.
  • the distance C2 between the centers of the recesses 68B is preferably 100 nm or more and 2000 nm or less, and more preferably 200 nm or more and 1000 nm or less, like the distance C1 between the centers of the recesses 68A.
  • the aspect ratio (H2 / D2) of the recess 68B is preferably 0.1 or more and 10 or less, more preferably 0.5 or more and 5.0 or less. is there.
  • the dimensions of the recess 68B are not limited to the above.
  • FIG. 8 is a diagram showing an AA cross section in FIG. 6 in the case of a convex portion.
  • the concave portions 68A and 68B as shown in FIG. 7 are non-flat portions, but the convex portions 68A ′ and 68B ′ may be non-flat portions as shown in FIG.
  • the protrusions 68 ⁇ / b> A ′ and 68 ⁇ / b> B ′ are columnar protrusions protruding from the surface 67.
  • FIG. 9 is a partially enlarged view of the wall surface of the recess.
  • the recess 68B is formed such that the wall surface 68A of the recess 68B has a predetermined angle with the bottom surface 68b of the recess 68B.
  • the metal film 66 provided on the surface 67 of the photonic crystal 65 is omitted.
  • the wall surface 68 ⁇ / b> A of the recess 68 ⁇ / b> B has a predetermined angle with respect to the bottom surface 68 b where the recess 68 ⁇ / b> B is flat.
  • the boundary between the wall surface 68A and the bottom surface 68b of the recess 68B is defined as a first boundary portion 71.
  • a boundary between the surface 67 and the wall surface 68 ⁇ / b> A of the recess 68 ⁇ / b> B is defined as a second boundary portion 72.
  • An intersection point between a straight line passing through the first boundary portion 71 in the vertical direction with respect to the bottom surface 68b and a straight line passing through the second boundary portion 72 in the horizontal direction with respect to the bottom surface 68b is defined as an intersection point A.
  • a distance connecting the first boundary 71 and the second boundary 72 with a straight line is L1.
  • a distance connecting the first boundary 71 and the intersection A with a straight line is L2.
  • a distance connecting the second boundary portion 72 and the intersection A with a straight line is L3.
  • ⁇ be the angle formed by L1 and L2.
  • an angle ⁇ formed by L1 and L2 is formed so as to satisfy the following expressions (1) and (2).
  • ⁇ peak is the peak wavelength
  • a 0 is the hole period
  • i, j are the diffraction orders
  • ⁇ n is the dielectric constant of the metal
  • ⁇ d is The dielectric constant of the environment.
  • the peak wavelength can be obtained if the period in which the recess 68B is disposed is given.
  • the position of the peak wavelength can be easily specified when the width of the spectrum of the peak wavelength is smaller. Therefore, when the period in which the recesses 68B are arranged is clearly given, the spectrum width of the peak wavelength is reduced, and the position of the peak wavelength is easily specified.
  • the metal film-coated photonic crystal 21 has a periodic structure in which the recesses 68B are periodically formed on the reflection surface 69.
  • the wall surface 68A of the recess 68B is formed on the reflection surface 69 so as to satisfy the above-described formulas (1) and (2), so that the width of the shape of the wavelength spectrum of the reflected light becomes narrow, and the peak wavelength of the reflected light Can be easily identified. Then, the target substance can be detected with high accuracy. As a result, the sensor sensitivity of the photonic crystal biosensor 11 can be improved.
  • the width of the shape of the wavelength spectrum of the reflected light is a half width.
  • the recess 68B is preferably formed so as to satisfy the following formula (2) ′.
  • the shape of the wavelength spectrum of the reflected light is further narrowed, and the peak wavelength of the reflected light is reduced. Can be identified more easily. As a result, the target substance can be detected with higher accuracy. 0 ⁇ tan ⁇ ⁇ 0.7 (2) ′
  • ⁇ is 0 degree or more.
  • the connection portion K between the surface 67 of the metal-coated photonic crystal 21 and the wall surface 68A of the recess 68B is approximately 90 degrees.
  • the connecting portion K is approximately 90 degrees, it becomes difficult to control the shape of the metal film-covered photonic crystal 21, particularly the shape of the recess 68B. That is, it is difficult to obtain the desired shape of the recess 68B.
  • the metal film-covered photonic crystal 21 is washed with water having a relatively high pressure.
  • the angle of the connection portion K is approximately 90 degrees, the corner is easily removed. As a result, the recess 68B may not have the desired shape. Since tan ⁇ > 0, that is, by making ⁇ larger than 0, the possibility that the corner of the connecting portion K can be taken can be reduced. Therefore, the concave portion 68B has a desired shape even after cleaning, which is preferable. Furthermore, since tan ⁇ > 0, that is, ⁇ is larger than 0, water can easily enter the recess 68B, so that the target substance can be reliably captured in the recess 29B.
  • FIG. 11 and FIG. 12 are diagrams for explaining a photonic crystal manufacturing method.
  • a process for producing the metal film-covered photonic crystal 21 by thermal nanoimprinting will be described.
  • a mold DI having a nanometer-level microstructure or a nanometer-level periodic structure pattern is used in thermal nanoimprint.
  • the heated mold DI is pressed against the sheet-shaped resin P, pressed at a predetermined pressure for a predetermined time, and released when the surface temperature of the mold DI reaches a predetermined temperature.
  • the structure and the periodic structure are transferred to the sheet-like resin P.
  • the photonic crystal 65 is obtained.
  • the mold DI is heated to about 160 ° C., pressed at a pressure of about 12 MPa for a predetermined time, and released when the surface temperature of the mold DI reaches about 60 ° C. It is preferable.
  • a metal film 66 is formed on the surface that has been in contact with the mold DI by sputtering or a vapor deposition apparatus to complete the metal film-covered photonic crystal 21. To do.
  • the target substance is an object to be detected by the target substance detection apparatus 10 and may be any of a polymer such as a protein, an oligomer, and a low molecule.
  • the target substance is not limited to a single molecule, and may be a complex composed of a plurality of molecules.
  • Examples of target substances include air pollutants, harmful substances in water, and biomarkers in the human body. Of these, cortisol and the like are preferable. Cortisol is a low molecular weight substance with a molecular weight of 362 g / mol.
  • Cortisol is attracting attention as a substance that evaluates the degree of stress felt by humans because cortisol concentration in saliva increases when humans feel stress.
  • concentration of cortisol as a target substance, for example, the degree of stress can be evaluated by measuring the concentration of cortisol contained in human saliva. If the degree of stress is evaluated, it can be determined whether or not the subject is in a stress state at a level that leads to mental illness such as depression.
  • the target substance capturing substance is a substance that binds to the target substance and captures the target substance.
  • the term “bonded” refers to a bond that is not chemically bonded, such as a bond by chemical adsorption or van der Waals force, in addition to the case of chemically bonding.
  • the target substance capturing substance is a substance that specifically reacts with the target substance to capture the target substance, and is preferably an antibody having the target substance as an antigen.
  • Specific reaction means selectively forming a complex by reversibly or irreversibly binding to a target substance, and is not limited to a chemical reaction.
  • a substance that reacts specifically may exist in addition to the target substance.
  • the target substance can be quantified if the affinity is very small compared to the target substance.
  • an antibody using the target substance as an antigen an artificially prepared antibody, a molecule composed of a substance constituting a DNA such as adenine, thymine, guanine, and cytosine, a peptide, and the like can be used.
  • the target substance capturing substance is preferably a cortisol antibody.
  • a known method can be employed to produce the target substance capturing substance.
  • the antibody can be produced by a serum method, a hybridoma method, or a phage display method.
  • Molecules composed of substances constituting DNA can be produced by, for example, the SELEX method (Systematic Evolution of Ligands by Exponential Enrichment).
  • the peptide can be prepared by, for example, a phage display method.
  • the target substance capturing substance does not need to be labeled with any enzyme / isotope. However, it may be labeled with an enzyme / isotope.
  • the target substance capturing substance is fixed to the reflection surface 69 of the metal film-coated photonic crystal 21 shown in FIG.
  • means for fixing the target substance capturing substance to the reflecting surface 69 of the metal film-coated photonic crystal 21 include chemical bonds such as covalent bonding, chemical adsorption, and physical adsorption, and physical bonding methods. These means can be appropriately selected according to the properties of the target substance-capturing substance. For example, when adsorption is selected as the fixing means, the adsorption operation is as follows.
  • a solution containing a target substance-capturing substance is dropped on the reflective surface 69 of the metal film-covered photonic crystal 21, and the metal film-covered photonic crystal 21 is cooled for a predetermined time at room temperature or as necessary.
  • the target substance trapping substance is adsorbed on the reflecting surface 69 by heating.
  • an antibody for example, cortisol antibody
  • a specific antigen for example, cortisol
  • the photonic crystal biosensor 11 can detect a specific antigen.
  • This utilizes the optical properties of the photonic crystal 65 and various biological / chemical reactions that occur on or near the surface of the photonic crystal 65, such as an antigen-antibody reaction in which a specific antigen reacts only with a specific antibody. To do.
  • the photonic crystal biosensor 11 may be one in which a blocking agent (protective substance) is immobilized on a reflective surface 69 on which an antibody that is a target substance capturing substance is immobilized.
  • the blocking agent is immobilized before the target substance is brought into contact with the photonic crystal biosensor 11.
  • the surface of the reflection surface 69 of the photonic crystal 65 is generally superhydrophobic. For this reason, impurities other than the antibody that is the target substance-capturing substance may be adsorbed on the reflecting surface 69 due to the hydrophobic interaction.
  • the optical characteristics of the photonic crystal 65 are greatly affected by the surface state, it is preferable that no impurities are adsorbed on the reflection surface 69 of the photonic crystal 65.
  • a so-called blocking agent should be fixed in advance so that impurities and the like are not fixed to portions other than the portion where the antibody that is the target substance capturing substance is adsorbed (fixed) to the reflection surface 69 of the photonic crystal 65. Is preferred.
  • the blocking agent In order to adsorb the blocking agent in advance, the blocking agent is brought into contact with the surface of the photonic crystal 65.
  • the blocking agent skim milk or bovine serum albumin (BSA) or the like can be used.
  • the photonic crystal biosensor 11 includes optical characteristics of the photonic crystal 65 and various biochemical reactions that occur on or near the surface of the photonic crystal 65, for example, a specific antigen is only a specific antibody. By utilizing the antigen-antibody reaction of reacting, a minute amount of protein or low molecular weight substance is detected.
  • the photonic crystal biosensor 11 reflects the wavelength of the reflected light due to the surface plasmon resonance phenomenon and / or the localized surface plasmon resonance phenomenon when the reflection surface 69 of the metal film-coated photonic crystal 21 is irradiated with light of a specific wavelength. Use the phenomenon of extreme values shifting.
  • an antibody (target substance-capturing substance) 74 is fixed to the surface of the reflection surface 69 of the metal film-coated photonic crystal 21 by adsorption.
  • a blocking agent (protective substance) 75 is preliminarily adsorbed on a portion of the reflective surface 69 other than the portion where the antibody 74 is adsorbed, that is, the reflective surface 69 other than the portion where the antibody 74 is adsorbed. . This prevents impurities or the like from being adsorbed on the reflective surface 69 other than the portion where the antibody 74 is adsorbed.
  • an antigen (target substance) 76 is brought into contact with the photonic crystal biosensor 11 on which the antibody 74 and the blocking agent 75 are adsorbed, and an antigen-antibody reaction is performed.
  • a complex 77 in which the antigen 76 is captured by the antibody 74 is fixed to the reflecting surface 69.
  • the light detection unit 12 shown in FIG. 1 converts light (incident light) LI having a specific wavelength into parallel light in a state where the antigen 76 is captured by the reflection surface 69 of the photonic crystal 65. Then, the reflective surface 69 of the metal film-coated photonic crystal 21 is irradiated. The light detection unit 12 illustrated in FIG. 1 detects the reflected light LR reflected by the reflecting surface 69, and obtains the wavelength of the extreme value of the reflected light LR. Then, the processing unit 13 shown in FIG.
  • the photonic crystal biosensor 11 changes the type of various biological substances such as proteins or low molecular weight substances, such as proteins, which are substances to be detected, by changing the type of combination of the antibody 74 and the antigen 76. Can do.
  • the photonic crystal biosensor 11 when the antigen 76 is captured by the antibody 74 fixed to the reflecting surface 69, the state of the reflecting surface 69 changes, and the reflected light LR changes.
  • the photonic crystal biosensor 11 outputs an optical physical quantity. This physical quantity correlates with a change in the surface state of the reflective surface 69 of the metal film-coated photonic crystal 21, and correlates with the amount of the complex 77 formed by capturing the antigen 76 on the antibody 74 fixed to the reflective surface 69. To do.
  • the optical physical quantity is, for example, the shift amount of the wavelength at which the intensity of the reflected light LR is an extreme value, the change amount of the reflectance of the light, the shift amount of the wavelength at which the reflectance of the light is an extreme value, the intensity of the reflected light LR. Or the amount of change in the extreme value of the intensity of the reflected light LR.
  • the shift amount of the wavelength at which the intensity of the reflected light LR or the reflectance of the light becomes an extreme value is used.
  • an optical physical quantity for example, it is performed as follows. Light is incident perpendicularly to the reflecting surface 69 of the metal film-covered photonic crystal 21, and the reflected light LR is detected. It is also possible to detect the reflected light LR by making light incident at an angle with respect to the normal of the reflecting surface 69 of the metal film-coated photonic crystal 21. By detecting the reflected light LR, the target substance detection device 10 shown in FIG. 1 can be made compact. In the case of detecting vertically incident and vertically reflected light, it is preferable to detect the reflected light LR by entering light using a bifurcated optical fiber. This structure will be described later.
  • FIG. 17 is a diagram showing the relationship between the intensity of the extreme value of reflected light and the wavelength.
  • FIG. 17 shows the reflected light intensity with respect to the wavelength (spectrum) of the reflected light.
  • FIG. 17B shows the relationship between the reflected light intensity and the wavelength when only the antibody 74 is adsorbed on the metal film 66 on the reflection surface 69 of the metal film-coated photonic crystal 21.
  • FIG. 17A shows the relationship between the reflected light intensity and the wavelength when the antigen 76 is captured by the antibody 74 fixed to the reflecting surface 69 of the metal film-coated photonic crystal 21.
  • the extreme values (minimum values) Pa and Pb of the reflected light intensity are taken when the wavelength is between 500 nm and 550 nm.
  • the wavelengths at that time are ⁇ b and ⁇ a ( ⁇ b ⁇ a).
  • the extreme value is greater than when only the antibody 74 is adsorbed on the metal film 66.
  • the wavelength of Pa shifts to a larger ⁇ a.
  • the target substance is detected using this wavelength shift amount (wavelength shift amount) ⁇ ( ⁇ a ⁇ b).
  • FIG. 18 is a diagram showing the relationship between the amount of wavelength shift at the extreme value of the intensity of reflected light and the concentration of avidin immobilized on the reflecting surface of the photonic crystal using biotin.
  • the result shown in FIG. 18 shows that the extreme value (minimum value) of reflected light intensity when biotin is immobilized as a target substance capturing substance on the reflection surface 69 of the metal film-coated photonic crystal 21 and avidin having a different concentration is dropped as the target substance. ) was obtained.
  • the wavelength shift amount ⁇ is a change amount (increase amount) from the wavelength in the extreme value (minimum value) of the reflected light intensity when the reflection surface 69 of the metal film-coated photonic crystal 21 is only the metal film 66. As shown in FIG.
  • the concentration DN of avidin as the target substance increases, and the wavelength shift amount ⁇ also increases.
  • the concentration of the target substance trapped on the reflection surface 69 of the metal film-covered photonic crystal 21 is obtained by obtaining the wavelength shift amount ⁇ .
  • the example described above is a case where biotin is used as a target substance capturing substance and avidin is used as a target substance, but the same result is obtained when cortisol is used as a target substance and a cortisol antibody is used as a target substance capturing substance.
  • the light detection unit 12 illustrated in FIG. 1 includes a light source 51, a measurement probe 52, a light detection device 53, a first optical fiber 54, a second optical fiber 55, and a collimator lens 56.
  • the light source 51 and the measurement probe 52 are optically connected by a first optical fiber 54.
  • the measurement probe 52 and the light detection device 53 are optically connected by a second optical fiber 55.
  • a control device that is connected to the light source 51 and the light detection device 53 and that controls the light source 51 and processes signals from the light detection device 53 may be provided.
  • the first optical fiber 54 shown in FIG. 1 guides the light from the light source 51 shown in FIG. 1 to the measurement probe 52, and the photonic crystal biosensor 11 included in the photonic crystal biosensor 11 from the measurement probe 52.
  • the reflective surface 69 of the crystal 21 is irradiated.
  • the collimating lens 56 emits the light emitted from the first optical fiber 54 and irradiated from the measurement probe 52 as parallel light, and then irradiates the reflecting surface 69 of the photonic crystal 65 as incident light LI.
  • the second optical fiber 55 receives the light reflected by the reflecting surface 69 of the metal film-covered photonic crystal 21 as reflected light LR and guides it to the light detection device 53 shown in FIG.
  • the light detection device 53 is a device for detecting light, which includes a light receiving element such as a phototransistor or a CCD (Charge Coupled Device).
  • FIG. 19 is a diagram showing a structure of a measurement probe included in the light detection unit shown in FIG.
  • the first optical fiber 54 and the second optical fiber 55 are joined.
  • the light exit surface 54P of the first optical fiber 54 and the incident surface 55P of the reflected light LR of the second optical fiber 55 are arranged on the same surface (incident / exit surface) 52P.
  • the measurement probe 52 includes the first optical fiber 54 and the second optical fiber 55 on the emission side (emission surface 54P side) of the first optical fiber 54 and the incident side (incident surface 55P side) of the second optical fiber 55. It is united. Then, the measurement probe 52 enters light using the first optical fiber 54 and the second optical fiber 55 and detects the reflected light LR.
  • the measurement probe 52 Since the measurement probe 52 has such a structure, the incident light LI irradiated to the reflection surface 69 of the photonic crystal 65 and the reflection light LR from the reflection surface 69 are emitted from substantially the same position and are incident. Can do. While the measurement probe 52 is configured as described above, and the collimator lens 56 is used to convert the light from the measurement probe 52 into parallel light, the light detection unit 12 converts the incident light LI of parallel light onto the reflection surface 69. It can be incident vertically. At the same time, the reflected light LR reflected perpendicularly from the reflecting surface 69 can be received.
  • the measurement probe 52 can suppress the fall of reflected light intensity to the minimum, and can mainly detect the 0th-order light component of the reflected light LR.
  • the processing unit 13 can obtain accurate information on the reflection surface 69 of the metal film-covered photonic crystal 21, so that the detection accuracy and concentration measurement accuracy of the target substance are improved.
  • the method for detecting the reflected light LR is not limited to the measurement probe 52 as described above.
  • a half mirror may be disposed between the collimating lens 56 and the reflection surface 69, and the reflected light LR may be separated by the half mirror and guided from the second optical fiber 55 to the light detection device 53.
  • the collimating lens 56 may be provided with an antireflection film.
  • the spectroscope includes a monochromator or a multi-channel spectroscope.
  • a multi-channel spectroscope is adopted from the viewpoint of a high detection speed.
  • a multi-channel spectroscope is a device that detects a spectrum by using photodetecting elements arranged in an array by dispersing incident light into a plurality of different wavelength regions using a prism, a grating, or the like.
  • the multichannel spectrometer can obtain a measurement result with a pitch of a specific wavelength width for each pixel of the photodetecting elements arranged in an array.
  • Pixel resolution is obtained by dividing the measurement range of one spectrometer by the number of pixels. Pixel resolution is the resolution of the wavelength of light that can be detected by the spectrometer.
  • CCD Charge Coupled Device
  • CMOS Complementary Metal Organic Semiconductor
  • any method may be used.
  • a photodiode, an avalanche photodiode, a photomultiplier tube, or the like may be arranged in an array.
  • the 20 and 21 are diagrams showing pixels of a spectroscope provided in the light detection device.
  • the spectroscope 53SA shown in FIG. 20 has five pixels D1, D2, D3, D4, and D5.
  • the spectroscope 53SB shown in FIG. 21 also includes five pixels D11, D12, D13, D14, and D114. Pixels D1, D2, D3, D4, and D5 of the spectroscope 53SA detect light having wavelengths ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4, and ⁇ 5 ( ⁇ 1 ⁇ 2 ⁇ 3 ⁇ 4 ⁇ 5), respectively.
  • Pixels D1, D2, D3, D4, and D5 of the spectroscope 53SB detect light having wavelengths ⁇ 11, ⁇ 12, ⁇ 13, ⁇ 14, and ⁇ 15 ( ⁇ 11 ⁇ 12 ⁇ 13 ⁇ 14 ⁇ 15), respectively.
  • the wavelengths ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4, and ⁇ 5 are increased by 1 ⁇ m in this order, and the wavelengths ⁇ 11, ⁇ 12, ⁇ 13, ⁇ 14, and ⁇ 15 are increased by 0.1 ⁇ m in this order. Therefore, the pixel resolution P1 of the spectroscope 53SA is 1 nm, and the pixel resolution P2 of the spectroscope 53SA is 0.1 nm.
  • the spectroscope 53SA and the spectroscope 53SB have the same number of pixels (number of pixels) (in the example shown in FIGS. 20 and 21, the number of pixels is the same for all five), the peak position of the spectrum is calculated. Therefore, a high spectroscope 53SB having pixel resolutions P1 and P2 of 0.1 nm is preferable because the shape of the spectrum peak can be accurately measured. Therefore, the pixel resolution is defined to be higher when the numerical value is smaller.
  • FIG. 22 is a diagram showing an example of a spectrum of reflected light detected by the spectrometer shown in FIG.
  • FIG. 23 is a diagram illustrating an example of a spectrum of reflected light detected by the spectroscope illustrated in FIG. 22 and 23, the horizontal axis represents the wavelength ⁇ , and the vertical axis represents the reflected light intensity.
  • the photodetection device 53 shown in FIG. 1 may include spectrometers 53SA and 53SB having different pixel resolutions, or may include a spectrometer having variable pixel resolution. In this way, an approximate peak position is determined by a wide measurement range (low pixel resolution), and an accurate peak position is detected by a narrow measurement range (high pixel resolution).
  • the photodetection device 53 includes the spectroscope 53SA and the spectroscope 53SB
  • an approximate peak position as shown in FIG. 22 is determined by the spectroscope 53SA having a wide measurement range, that is, a relatively low pixel resolution P1.
  • An accurate peak position as shown in FIG. 23 is detected by the spectroscope 53SB having a narrow measurement range, that is, a relatively high pixel resolution P2.
  • the processing unit 13 obtains the wavelength of the extreme value of the reflected light using the spectroscope 53SA as the first spectroscope, and then sets the spectroscope 53SB as the second spectroscope.
  • the wavelength of the extreme value of the reflected light is obtained within the range of the wavelength of the extreme value obtained by the spectroscope 53SA. By doing so, it is possible to achieve both a wide measurement range and accurate peak position detection.
  • FIG. 24-1 is a diagram illustrating an example of a spectrum of reflected light when the light detection element provided in the spectroscope is not cooled.
  • FIG. 24-2 is a diagram illustrating an example of a spectrum of reflected light when the light detection element included in the spectroscope is cooled.
  • the horizontal axis represents the wavelength ⁇
  • the vertical axis represents the reflected light intensity.
  • the dotted line ST is the spectrum of the actual reflected light
  • the solid line SG is the detection result of the spectrum of the reflected light detected by the spectrometer.
  • the light detection element In order to remove this noise, it is preferable to cool the light detection element. By cooling the light detection element, noise caused by heat can be reduced as in the detection result SG indicated by the solid line in FIG. In order to cool the light detection element, for example, a Peltier element or the like can be used.
  • FIG. 25A is a diagram illustrating an example of a spectrum of reflected light detected by a light detection element included in the spectroscope.
  • FIG. 25-2 is a diagram for explaining an example of obtaining the peak position by data fitting the result shown in FIG. 25-1.
  • the vertical axis represents the reflectance
  • the horizontal axis represents the wavelength ⁇ .
  • the pixel resolution of the spectroscopes 53SA, 53SB, etc. is higher.
  • the peak position PK can be obtained with resolution.
  • the wavelength of the reflected light at the peak position PK corresponds to the wavelength of the reflected light at the extreme value. That is, the peak position PK is an extreme value position. If the peak position PK can be accurately determined, the wavelength of the reflected light at the extreme value can also be accurately determined.
  • an n-order function (n is a natural number of 2 or more), a Lorentz function, a Gaussian function, a Forked function, a beta function, or the like, or a function obtained by combining a plurality of these functions can be used.
  • FIG. 25-3 and FIG. 25-4 are diagrams showing the peak position obtained from the detection result of the light detection device and the peak position obtained by fitting the detection result data of the light detection device.
  • the detection result of the light detection device 53 is indicated by black dots in FIGS. 25-3 and 25-4.
  • FIG. 25C when the peak position obtained from the detection result of the light detection device 53 is obtained, the position PKr1 is obtained.
  • FIG. 25-3 it is estimated that there is a peak position between the position PKr1 and the position PKr2 from the change in the detection result.
  • the peak position PKf obtained by fitting the detection results of the light detection device 53, the spectroscopes 53SA and 53SB in the present embodiment, and the peak fitting in the present embodiment exists between the position PKr1 and the position PKr2.
  • a resolution higher than the pixel resolution of the spectroscopes 53SA, 53SB and the like can be realized, so that the more probable peak position PKf, that is, the wavelength of the reflected light at the extreme value can be obtained. it can.
  • the extreme value of the reflected light can be obtained. What is necessary is just to obtain
  • the position PKr when the peak position obtained from the detection result of the light detection device 53 is obtained, the position PKr is obtained.
  • the position PKr includes an error due to a change in the detection result.
  • a resolution higher than the pixel resolution of the spectroscopes 53SA, 53SB and the like can be realized, so that a more probable peak position PKf, that is, the wavelength of reflected light at the extreme value can be obtained.
  • FIG. 25-5 is a diagram showing the change with time of the peak wavelength obtained from the detection result of the photodetection device.
  • FIG. 25-6 is a diagram showing a change with time of the peak wavelength obtained by peak fitting the detection result of the photodetection device. As shown in these figures, it can be seen that the time change of the peak wavelength ⁇ p obtained by the peak fitting becomes smoother than the time change of the peak wavelength ⁇ p obtained from the detection result of the light detection device 53.
  • FIG. 25-7 is a flowchart showing each process of peak fitting.
  • the peak fitting is executed by the processing unit 13 of the target substance detection apparatus 10 shown in FIG.
  • step S ⁇ b> 1 the processing unit 13 acquires the detection result of the light detection device 53 and extracts the detection result that takes the minimum value from the detection result group around the bottom peak.
  • step S2 the processing unit 13 extracts detection results up to ⁇ nth on the basis of the detection result having the minimum value.
  • n is an integer greater than 1.
  • step S3 the processing unit 13 performs peak fitting using 2 ⁇ n + 1 detection results. For peak fitting, the above-described arbitrary function is used.
  • step S4 the processing unit 13 calculates a residual between the curve obtained by the peak fitting and the detection result of the light detection device 53, and the calculated residual is smaller than a predetermined set value. (Step S4, Yes), the process proceeds to Step S5.
  • step S5 the processing unit 13 estimates a peak position from a fitting function, that is, a function used for peak fitting.
  • step S4 when the calculated residual is equal to or greater than a predetermined set value (step S4, No), the process proceeds to step S6.
  • step S6 the processing unit 13 changes at least one of the fitting function and the initial parameter, and executes step S3 and step S4.
  • the above-mentioned n-order function (n is a natural number of 2 or more) exemplified as the fitting function, Lorentz function, Gaussian function, Forked function, beta function, etc. are not superior or inferior.
  • the function having the shape closest to the detection result group to be peak-fitted, that is, the function having the shape having the smallest residual is the most suitable function in that case.
  • a liquid handling unit 14 shown in FIG. 1 discharges from a first container 30 that holds a liquid L such as a solution containing a target substance-capturing substance, a pump 31 as a liquid feeding device, and a photonic crystal biosensor 11. And a second container 32 for storing the liquid L.
  • the pump 31 is controlled by the processing unit 13 shown in FIG.
  • a liquid supply pipe 25 is inserted into the first container 30.
  • the liquid discharge pipe 26 is connected to the inlet of the pump 31.
  • a discharge pipe 33 connected to the outlet of the pump 31 is inserted into the second container 32.
  • the pump 31 sucks the liquid L from the liquid discharge pipe 26 to supply the liquid L in the first container 30 into the opening 23P of the photonic crystal biosensor 11.
  • the pump 31 sucks the liquid L in the opening 23P of the photonic crystal biosensor 11 and discharges it from the discharge pipe 33 to the second container 32.
  • the liquid handling unit 14 supplies the liquid L such as a solution containing the target substance capturing substance into the opening 23P of the photonic crystal biosensor 11 by the pump 31.
  • FIG. 26 and FIG. 27 are diagrams showing modifications of the liquid handling unit.
  • the liquid handling unit 14a illustrated in FIG. 26 includes a first container 30A, a pump 31, a second container 32, a three-way valve 34, and a third container 31B.
  • a first liquid supply pipe 25A and a second liquid supply pipe 25B are connected to the first inlet 34I and the second inlet 34I2, which are the two inlets of the three-way valve 34, respectively.
  • the first liquid supply pipe 25A is inserted into the first container 30A, and the second liquid supply pipe 25B is inserted into the second container 30B.
  • a liquid supply pipe 25 is connected to the outlet 34E of the three-way valve 34.
  • the three-way valve 34 can switch between a first state in which the first inlet 34I1 and the outlet 34E are connected and a second state in which the second inlet 34I2 and the outlet 34E are connected.
  • the control device 13 shown in FIG. 1 controls the three-way valve 34 to switch between the first state and the second state.
  • the liquid handling part 14a can simplify the work of supplying a plurality of liquids L into the opening 23P of the photonic crystal biosensor 11.
  • the liquid handling part 14b shown in FIG. 27 is the same as the liquid handling part 14 shown in FIG. 1, but the liquid handling part 14 causes the liquid L to flow into the opening 23P of the photonic crystal biosensor 11 by negative pressure (negative pressure). Is different from that supplied by positive pressure (positive pressure). For this reason, the suction pipe 35 connected to the inlet of the pump 31 is inserted into the first container 30 ⁇ / b> A, and the liquid supply pipe 25 is connected to the outlet of the pump 31. The liquid discharge pipe 26 is inserted into the second container 30B. When the pump 31 is driven, the liquid L sucked from the first container 30A by the pump 31 is discharged from the outlet of the pump 31, and then passes through the liquid supply pipe 25 to open the opening 23P of the photonic crystal biosensor 11. Supplied in.
  • [Modification of photonic crystal biosensor] 28, 29 and 30 are diagrams showing a first modification of the photonic crystal biosensor.
  • the photonic crystal biosensor 11A has the same structure as the photonic crystal biosensor 11 described above, but the support member 24A is placed between the support member 24A and the metal film-coated photonic crystal 21. The difference is that it has a plurality of claws 41 that engage with the holding member 23 sandwiching the metal film-coated photonic crystal 21.
  • Other structures are the same as those of the photonic crystal biosensor 11 described above. As shown in FIGS.
  • the plurality of claws 41 are provided on the surface of the support member 24 ⁇ / b> A on which the metal film-coated photonic crystal 21 is placed, and on the tip of the support body 40 provided on the outer edge thereof. ing. As shown in FIG. 30, the claw 41 has a triangular cross section.
  • the holding member 23 and the covering member 22 are fitted between the plurality of claws 41 in this order.
  • the claw 41 engages with the surface of the covering member 22, thereby sandwiching the metal film-coated photonic crystal 21 between the holding member 23 and the support member 24 via the covering member 22 and the holding member 23.
  • the photonic crystal biosensor 11A can fix the metal film-coated photonic crystal 21 without using the mounting jigs 27 and 28 shown in FIG. As shown in FIG. 30, the distance between the facing claws 41 increases as the claw 41 moves away from the support member 24 ⁇ / b> A. By doing so, the holding member 23 and the covering member 22 can be easily fitted into the support member 24A on which the metal film-covered photonic crystal 21 is placed.
  • FIGS. 31 and 32 are diagrams showing a second modification of the photonic crystal biosensor.
  • the photonic crystal biosensor 11B has the same structure as the photonic crystal biosensor 11 described above, but the support member 24B holds the holding member 23 on the side on which the metal film-coated photonic crystal 21 is placed, And the point provided with the part which engage
  • Other structures are the same as those of the photonic crystal biosensor 11 described above.
  • a wall 24P on which the metal film-coated photonic crystal 21 is placed on the support member 24A, and a wall on which the holding member 23 is held and the covering member 22 is fitted on the outer edge thereof. 42 is provided.
  • the wall 42 rises in the thickness direction of the support member 24 from the outer edge portion of the surface 24P.
  • the wall 24 surrounds a surface 24P on which the metal film-coated photonic crystal 21 of the support member 24A is placed.
  • the metal film-covered photonic crystal 21 is placed on the portion surrounded by the wall 42.
  • the holding member 23 By attaching the holding member 23 to a portion surrounded by the wall 24, the metal film-covered photonic crystal 21 is sandwiched between the holding member 23 and the support member 24B.
  • the covering member 22 is attached to the surface of the holding member 23.
  • the dimension of the portion surrounded by the wall 42 is smaller than the dimension of the outer edge of the covering member 22. For this reason, the covering member is fixed to the wall 42 by being fitted into the wall 42.
  • the photonic crystal biosensor 11B can fix the metal film-covered photonic crystal 21 without using the mounting jigs 27 and 28 shown in FIG.
  • a liquid is introduced into the opening 23P of the holding member 23 such as the photonic crystal biosensor 11 or the like.
  • the liquid in the opening 23P can be replaced while the metal film-coated photonic crystal 21 is sandwiched between the support member 24 and the holding member 23.
  • measurement noise due to errors in attaching the metal film-covered photonic crystal 21 can be reduced.
  • the detection sensitivity of the target substance is improved.
  • the configurations of the present embodiment and its modifications can be applied or combined as appropriate in the following embodiments.
  • FIG. 33 and 34 are diagrams showing a photonic crystal biosensor according to the second embodiment.
  • the second embodiment is different from the first embodiment and the modification thereof in that the holding member includes a plurality of openings. Since other structures are the same as those of the first embodiment and the modifications thereof, the description of the same parts is omitted as necessary.
  • the photonic crystal biosensor 11C includes a support member 24C, a holding member 23C, and a covering member 22.
  • the holding member 23C has a plurality of openings 23P1, 23P2, and 23P3 that overlap the portion 21C that captures the target substance of the metal film-coated photonic crystal 21 placed on the support member 24C. As shown in FIG.
  • the openings 23P1, 23P2, and 23P3 pass through two largest opposing flat surfaces of the holding member 23C that is a plate-like member.
  • the openings 23P1, 23P2, and 23P3 are groove-shaped as shown in FIG. 34, and are opened so as to overlap the portion 21C that captures the target substance of the metal film-coated photonic crystal 21.
  • the plurality of openings 23P1, 23P2, and 23P3 do not intersect each other.
  • the respective openings 23P1, 23P2, and 23P3 are arranged in parallel to the portion 21C that captures the target substance of the metal film-coated photonic crystal 21.
  • the plurality of openings 23P1, 23P2, and 23P3 do not need to be parallel to each other.
  • the support member 24C on which the metal film-coated photonic crystal 21 is placed has a plurality of holes 24I1, 24I2, 24I3, 24E1, 24E2, and 24E3.
  • the openings 23P1, 23P2, and 23P3 also overlap with the holes 24I1, 24I2, 24I3, 24E1, 24E2, and 24E3 of the support member 24C.
  • the holes 24I1, 24I2, 24I3, 24E1, 24E2, and 24E3 are one of the plurality of openings 23P1, 23P2, and 23P3 in a state where the metal film-coated photonic crystal 21 is sandwiched between the holding member 23 and the support member 24. Two of them are open to the respective openings 23P1, 23P2, 23P3.
  • the hole 24I1 and the hole 24E1 open in the opening 23P1, the hole 24I2 and the hole 24E2 open in the opening 23P2, and the hole 24I3 and the hole 24E3 open in the opening 23P3.
  • the hole 24I1 supplies a liquid such as a solution containing a target substance-capturing substance into the opening 23P1, the hole 24I2 into the opening 23P2, and the hole 24I3 into the opening 23P3.
  • the hole 24E1 discharges a liquid such as a solution containing a target substance-trapping substance from the opening 23P1, the hole 24E2 from the opening 23P2, and the hole 24E3 from the opening 23P3.
  • the holes 24I1, 24I2, and 24I3 are appropriately referred to as liquid supply holes 24I1, 24I2, and 24I3, and the holes 24E1, 24E2, and 24E3 are referred to as liquid discharge holes 24E1, 24E2, and 24E3.
  • the photonic crystal biosensor 11C can introduce a liquid into each of the openings 23P1, 23P2, and 23P3, so that one metal film-coated photonic crystal 21 evaluates different types of liquids. You can also
  • the liquid discharge holes 24E1, 24E2, 24E3 are connected to the inlets of the pumps, and the liquid is introduced into the openings 23P1, 23P2, 23P3 using negative pressure.
  • a pump may be provided corresponding to each of the openings 23P1, 23P2, and 23P3, or a single pump supplies liquid to each of the openings 23P1, 23P2, and 23P3, and each of the openings 23P1.
  • 23P2 and 23P3 may discharge the liquid.
  • the pump outlet may be connected to the liquid supply holes 24I1, 24I2, and 24I3, and the liquid may be introduced into the openings 23P1, 23P2, and 23P3 using positive pressure.
  • pumps are provided corresponding to the respective openings 23P1, 23P2, and 23P3.
  • the metal film-coated photonic crystal 21 has a fine structure, it is difficult to precisely control the shape even if it is manufactured by the same manufacturing process. For this reason, variation exists for each metal film-coated photonic crystal 21. Since the photonic crystal biosensor 11C can introduce a liquid into each of the openings 23P1, 23P2, and 23P3, the metal film-covered photonic crystal 21 can be calibrated simultaneously with the inspection. As a result, the photonic crystal biosensor 11C can realize highly accurate measurement. For example, a solution to be inspected and a standard solution having a known property (for example, concentration) are simultaneously introduced into the portion 21C for capturing the target substance of the metal film-coated photonic crystal 21.
  • a solution to be inspected and a standard solution having a known property for example, concentration
  • the metal film-covered photonic crystal 21 can be calibrated simultaneously with the inspection by obtaining a calibration curve from the detection result of the standard solution and the detection result of the solution to be inspected.
  • the photonic crystal biosensor 11C can measure the concentration of the target substance contained in the solution to be examined with high accuracy.
  • the volume of the space surrounded by each opening 23P1, 23P2, 23P3, the support member 24C, and the covering member 22 is the volume of the space surrounded by the opening 23P, the supporting member 24, and the covering member 22 in the first embodiment. Since the volume is smaller than the volume, the amount of liquid supplied to the openings 23P1, 23P2, and 23P3 is small. For this reason, it is particularly preferable when an expensive liquid is used.
  • the holding member 23C has two or more openings, the metal film-covered photonic crystal 21 can be calibrated. For this reason, the holding member 23C preferably has two or more openings. In order to calibrate the metal film-coated photonic crystal 21 more accurately, it is preferable to introduce a plurality of standard solutions in addition to the solution to be inspected. For this reason, it is more preferable that the holding member 23C has three or more openings.
  • FIG. 35 is a perspective view showing a light detection unit according to the second embodiment.
  • 36 and 37 are exploded views of the light detection unit according to the second embodiment.
  • the light detection unit 50 is a portion that captures a target substance of the metal film-coated photonic crystal 21 from a plurality of (three in this embodiment) openings 23P1, 23P2, and 23P3 provided in the holding unit 23C of the photonic crystal biosensor 11C. 21C is irradiated with light and reflected light is received. For this reason, as shown in FIGS. 35 and 36, the light detection unit 50 includes a plurality of measurement probes 52C.
  • a plurality of measurement probes 52C are stored in a housing 43.
  • the housing 43 is divided into a first housing 43A and a second housing 43B.
  • a holding unit 44 that houses and holds a plurality of measurement probes 52 ⁇ / b> C is attached inside the housing 43.
  • the holding unit 44 is divided into a first member 44A and a second member 44B.
  • a plurality of measurement probes 52C are arranged between the first member 44A and the second member 44B.
  • the holding unit 44 there are a plurality of (three in this embodiment) openings 46 for irradiating light to the portion 21C that captures the target substance of the metal film-coated photonic crystal 21 and receiving reflected light. Is provided.
  • the intervals between the plurality of openings 46 are the same as the intervals between the plurality of openings 23P1, 23P2, and 23P3 in the portion 21C that captures the target substance of the metal film-coated photonic crystal 21.
  • the measurement probe 52 ⁇ / b> C is formed by joining the first optical fiber 54 and the second optical fiber 55, and the light emission surface of the first optical fiber 54 and the incident surface of the second optical fiber 55. It arrange
  • a collimator lens 56 ⁇ / b> C is disposed between the incident / exit surface 52 ⁇ / b> P of the measurement probe 52 and the opening 46.
  • the opening 46 is a combination of a notch 46A formed at one end of the first member 44A and a notch 46B formed at one end of the second member 44B.
  • the second member 44B has a plurality of grooves 45 for holding the measurement probe 52C.
  • the first member 44A also has a plurality of grooves 45.
  • the measurement probe 52C is sandwiched and held in the groove 45 of the first member 44A and the second member 44B.
  • the collimating lens 56C is a spherical lens. As shown in FIG. 37, the second member 44B has a plurality of recesses 47 for holding the collimating lens 56C. Similarly to the second member 44B, the first member 44A also has a plurality of recesses 47. The measurement probe 52C is sandwiched and held in the recesses 47 of the first member 44A and the second member 44B. With such a structure, the light detection unit 50 irradiates light to the portion 21C that captures the target substance of the metal film-coated photonic crystal 21 through the adjacent openings 23P1, 23P2, and 23P3 adjacent to each other. Reflected light can be received.
  • the liquid can be introduced into the respective openings 23P1, 23P2, and 23P3 provided in the photonic crystal biosensor 11C, the metal film-covered photonic crystal 21 can be calibrated simultaneously with the inspection. .
  • this embodiment can realize highly accurate measurement.
  • the volume of the space surrounded by the openings 23P1, 23P2, 23P3, the support member 24C, and the covering member 22 is small, the amount of liquid supplied to the openings 23P1, 23P2, 23P3 can be small.
  • FIG. 38 is a diagram showing a target substance detection device.
  • a target substance detection apparatus provided with a target substance capturing apparatus according to Embodiment 3 will be described.
  • the target substance detection device 10c includes a photonic crystal biosensor (target substance capture device) 11 according to the third embodiment, a light detection unit 12, and a control unit 13c.
  • the photonic crystal biosensor 11c includes a metal film-coated photonic crystal 21, a table 83, a thin plate 84, and a cover 82.
  • the photonic crystal biosensor 11c has a structure in which the metal film-covered photonic crystal 21 is disposed in a flow path 84f formed by the base 83, the thin plate 84, and the cover 82. Since the metal film-covered photonic crystal 21 is the same as that of the embodiment mobile phone 1, the description thereof is omitted.
  • FIG. 39 is an explanatory diagram of the photonic crystal biosensor 11c.
  • the photonic crystal biosensor 11c includes a metal film-coated photonic crystal 21, a table 83 having two through holes 83h, a thin plate 84 having an opening 84h, and a cover 82.
  • the metal film-coated photonic crystal 21 is installed on the surface of the table 83. Thereafter, the thin plate 84 is placed on the table 83.
  • the width of the metal film-covered photonic crystal 21 is smaller than the width of the opening 84h.
  • the metal film-covered photonic crystal 21 is sandwiched and fixed between the base 83 and the thin plate 84.
  • the cover 82 is installed on the thin plate 84.
  • the photonic crystal biosensor 11c has a channel 83f formed by being surrounded by the base 83, the inner wall of the thin plate 84 on the opening 84h side, and the cover 82.
  • the inner wall on the opening 84h side refers to the inner wall of the thin plate 84, which is a boundary surface between the thin plate 84 and the opening 84h.
  • the metal film-covered photonic crystal 21 is disposed in the flow path 84f.
  • the solution containing the target substance flows through the flow path 84f, so that the reflection surface 69 captures the target substance.
  • the channel 84f may not be formed as described above.
  • the channel 84f may be formed by recessing a part of the surface of the table 83.
  • the photonic crystal biosensor 11 c includes a supply pipe 96 and a discharge pipe 97.
  • the solution is supplied from the supply pipe 96 to the flow path 84f.
  • the solution is discharged from the flow path 84f through the discharge pipe 97.
  • the material of the base 83 and the cover 82 is not particularly limited. However, in consideration of the cleanliness of the surfaces of the cover 82 and the base 83, it is preferably formed using stainless steel, polycycloolefin resin, silica, or the like.
  • One of the two through holes 83h is a supply port for allowing the solution to flow into the flow path 84f.
  • the other of the two through holes 83h is a discharge port through which the solution flows out from the flow path 84f.
  • a supply pipe 96 having a connector 79 at the tip is connected to one of the two through holes 83h.
  • a discharge pipe 97 having a connector 79 at the tip is connected to the other of the two through holes 83h.
  • the solution flows into the flow path 84f through the supply pipe 96 and flows out of the flow path 84f through the discharge pipe 97. Further, the connector 79 closes the two through holes 83h. For this reason, the connector 79 reduces the possibility that the solution leaks from the flow path 84f.
  • the through hole 83h, the supply pipe 96, and the discharge pipe 97 may not be provided. Even when the through hole 83h, the supply pipe 96, and the discharge pipe 97 are not provided, for example, if the flow path 84f is formed in an annular shape, the solution circulates through the flow path 84f. Further, there may be three or more through holes 83h.
  • the photonic crystal biosensor 11c is uniformly produced by thermal nanoimprint or the like. In order for the target substance detection device 10c to detect the reflected light more accurately, it is preferable to accurately position the incident site and the reflected site of the light irradiated to the photonic crystal biosensor 11c.
  • the positional relationship at the time of measurement between the photonic crystal biosensor 11c and the measurement probe described later is preferably the same before and after the antigen-antibody reaction, and the same part is preferably measured. Therefore, the distance between the measurement probe and the reflection surface 69 of the photonic crystal biosensor 11c is preferably the same before and after the antigen-antibody reaction, and is preferably fixed to 50 ⁇ m to 500 ⁇ m. Since the photonic crystal biosensor 11c includes the cover 82, the cover 82 functions as a spacer, and the distance between the measurement probe and the reflection surface 69 of the photonic crystal biosensor 11c can be made constant.
  • the photonic crystal biosensor 11c may be marked with a positioning marker that displays a specific position on the reflecting surface 69.
  • the marker can be attached by photolithography, sputtering, vapor deposition, a lift-off process using these, printing with ink or the like, or pattern formation by imprinting.
  • the marker may be attached to either the front surface (the reflective surface 69 side) or the back surface (the opposite side of the reflective surface 69) of the photonic crystal biosensor 11c as long as the position can be read.
  • the measurement part of the photonic crystal 65 may be removed and a marker may be attached to the photonic crystal 65 itself.
  • a marker may be attached to the cover 82 and the base 83.
  • FIG. 40 is a diagram illustrating a state before the solution is supplied to the flow path 84f.
  • FIG. 41 is a diagram showing a state in which a solution is circulated.
  • the photonic crystal biosensor 11 c includes a pump 91, a valve 94, a supply pipe 95, a discharge pipe 98, a container 92, and a new solution 93.
  • the supply pipe 96 is connected to the supply pipe 95 via a passage 94 a inside the valve 94.
  • An end portion 95 e of the supply pipe 95 is immersed in a new solution 93 stored in the container 92.
  • the discharge pipe 97 is connected to the discharge pipe 98 via a passage 94 b inside the valve 94.
  • the control unit 13c is connected to the valve 94 and can switch the direction in which the solution is guided.
  • the pump 91 is provided in the discharge pipe 97.
  • the pump 91 exhibits a function of applying a negative pressure to the flow path 84f.
  • the pump 91 may be provided in the supply pipe 96. When the pump 91 is provided in the supply pipe 96, the pump 91 exhibits a function of applying a positive pressure to the flow path 84f.
  • FIG. 42 is a flowchart illustrating an example of a solution circulation method according to the third embodiment.
  • a method for circulating the solution will be described.
  • step S11 the solution stored in the container 92 by the pump 91 is sent to the flow path 84f.
  • the pump 91 is activated, a negative pressure is applied to the flow path 84f.
  • pressure is also transmitted to the supply pipes 96 and 95 connected to the flow path 84f, and the new solution 93 stored in the container 92 is sucked up from the end portion 95e of the supply pipe 95.
  • the solution flows into the channel 84f.
  • the solution passes through the space 21 u above the reflecting surface 69 and reaches the passage 94 b through the discharge pipe 97.
  • step S12 the valve 94 is switched to circulate the solution.
  • the control unit 13c switches the valve 94.
  • the supply pipe 96 and the discharge pipe 97 are connected via the passage 94b.
  • the solution circulates through the supply pipe 96, the flow path 84 f and the discharge pipe 97.
  • the solution that has passed through the channel 84f is repeatedly guided to the channel 84f.
  • the solution that has passed through the space 21 u above the reflecting surface 69 is repeatedly guided to the space 21 u above the reflecting surface 69.
  • the reflecting surface 69 can repeatedly obtain the opportunity to capture the target substance that has passed through the space 21u above the reflecting surface 69 without being captured.
  • step S13 after the measurement of the reflected light is completed, the end portion 95e of the supply pipe 95 is lifted from the new solution 93 stored in the container 92, and the valve 94 is switched.
  • the valve 94 is switched, the discharge pipe 97 is connected to the discharge pipe 98 via the passage 94b.
  • the solution inside the flow path 84f, the supply pipe 96 and the discharge pipe 97 is discharged from the end portion 98e of the discharge pipe 98.
  • the method for circulating the solution may not be the above method.
  • FIG. 43 is an explanatory diagram of another circulation method.
  • the solution circulation method may be a method using the configuration shown in FIG.
  • the end 96 e of the supply pipe 96 and the end 97 e of the discharge pipe 97 are immersed in a new solution 93 stored in the container 92.
  • a negative pressure is applied to the flow path 84f. Therefore, the pressure is also transmitted to the supply pipe 96 connected to the flow path 84f, and the new solution 93 stored in the container 92 is sucked up from the end portion 96e of the supply pipe 96. As a result, the solution flows into the channel 84f.
  • the pump 91 may be provided in the supply pipe 96. In this case, the pump 91 exhibits a function of applying a positive pressure to the flow path 84f.
  • the solution that has passed through the space 21u above the reflecting surface 69 is repeatedly guided to the space 21u above the reflecting surface 69.
  • the solution that has passed through the space 21u above the reflecting surface 69 without reacting with the metal film-covered photonic crystal 21 can be repeatedly obtained with the opportunity to react with the metal film-covered photonic crystal 21. it can.
  • increasing the flow rate of the solution does not increase the amount of solution required to reach equilibrium. Therefore, the photonic crystal biosensor 11c according to the third embodiment reduces the amount of solution required to reach the equilibrium state while speeding up the change in the reflected light of the light applied to the metal film-coated photonic crystal 21. it can.
  • the solution is supplied from the supply pipe 96 to the flow path 84f.
  • the solution is discharged from the flow path 84f through the discharge pipe 97.
  • the pump 91 for moving a solution can be installed in the exterior of the flow path 84f. Since the flow path 84f is very small, when the pump 91 can be installed outside the flow path 84f, the assembly of the photonic crystal biosensor 11c becomes easy. Therefore, the photonic crystal biosensor 11c according to the third embodiment is easy to assemble, and reaches the equilibrium state while further rapidly changing the reflected light of the light applied to the metal film-coated photonic crystal 21. The amount of solution required can be reduced.
  • the photonic crystal biosensor 11c according to the third embodiment has a channel 83f formed by being surrounded by the base 83, the inner wall of the thin plate 84 on the opening 84h side, and the cover 82.
  • the flow path 84f can be formed thin, and the flow rate of the solution passing through the space 21u above the reflection surface 69 can be increased.
  • the target substance is quickly captured by the reflecting surface 69. Therefore, the photonic crystal biosensor 11c according to the third embodiment can reduce the amount of solution required to reach an equilibrium state while further rapidly changing the reflected light of the light applied to the metal film-coated photonic crystal 21. Less.
  • An Example is the result of the experiment which performed the real-time measurement of reflected light using the circulation method mentioned above.
  • the example is the result of an experiment in which the solution was circulated through the flow path 84f so that the flow rate per unit time was 300 ⁇ l / min and the Reynolds number was 4.0.
  • the amount of the solution used for flowing the flow path 84f is 1.5 ml.
  • the comparative example is the result of an experiment in which reflected light was measured in real time in a state where the solution was allowed to stand without flowing.
  • biotin was immobilized on the reflective surface 69 and reacted with 100 nM avidin.
  • FIG. 44 is a diagram showing a change in the wavelength of the extreme value of reflected light with respect to time in the example and the comparative example.
  • the solution begins to contact the reflective surface 69 from time Ts. Comparing the example and the comparative example, it can be seen that the example reacts more quickly. Therefore, the photonic crystal biosensor 11c according to the third embodiment reduces the amount of solution required to reach the equilibrium state while speeding up the change in the reflected light of the light applied to the metal film-coated photonic crystal 21. it can.
  • the relationship between the flow rate of the solution and the cross-sectional shape of the flow path 84f is preferably such that the Reynolds number is 0.01 or more and 2000 or less. This is because, when the Reynolds number is 2000 or less, a turbulent flow component is hardly generated, and thus the possibility that noise is generated in the measurement result of reflected light is reduced. Further, when the Reynolds number is 2000 or less, it is difficult to apply a large pressure to the flow path 84f, so that the possibility that the solution leaks from the flow path 84f is reduced. Furthermore, the relationship between the flow rate of the solution and the cross-sectional shape of the flow path 84f is preferably a relationship in which the Reynolds number is 0.01 or more and 1000 or less. This is because when the Reynolds number is 1000 or less, a stable laminar flow is likely to occur, so that the possibility of noise occurring in the measurement result of reflected light is further reduced.
  • FIG. 45 is a diagram illustrating evaluation conditions of the light detection unit 12 of the target substance detection device 10c according to the third embodiment. Next, the evaluation conditions of the light detection unit 12 will be described.
  • the light detection unit 12 arranges a collimator lens 56 between the incident / exit surface 63 of the measurement probe 52 and the reflection surface 69 of the metal film-covered photonic crystal 21.
  • the distance (measurement distance) between the collimating lens 56 and the reflecting surface 69 is h
  • the diameter of the parallel light emitted from the collimating lens 56 is d1
  • the diameter of the portion where the reflecting surface 69 of the photonic crystal 65 is exposed is d2.
  • h was 15 mm or 40 mm
  • d1 was 3.5 mm
  • d2 was 5 mm.
  • the optical axis ZL of the light applied to the reflecting surface 69 and the optical axis ZL of the reflected light reflected by the reflecting surface 69 are both orthogonal to the reflecting surface 69.
  • the diameter of the measurement probe 52 is 200 ⁇ m.
  • White light was used as the irradiation light.
  • the reflectance is the ratio of the standard material (aluminum plate) to the reflected light intensity.
  • control unit 13c obtains the wavelength of the extreme value of the reflected light detected by the light detection unit 12. At the same time, the control unit 13c detects at least the presence or absence of the target substance (for example, the antigen 76 shown in FIGS. 11, 12, etc.) based on the obtained wavelength shift (wavelength shift amount) of the extreme value.
  • the control unit 13c is, for example, a microcomputer. There is a correlation between the amount of wavelength shift and the concentration of the target substance trapped on the reflection surface 69 of the metal film-coated photonic crystal 21.
  • control part 13c can obtain
  • the control unit 13c is connected to the valve 94.
  • the controller 13c switches the valve 94 based on the state of the passage 94b inside the valve 94.
  • a method for detecting a target substance using the target substance detection apparatus 10 shown in FIG. 1 and the target substance detection apparatus 10c shown in FIG. 38 will be described.
  • a case will be described in which cortisol antibody is adsorbed on the reflective surface 69 of the metal film-coated photonic crystal 21 and cortisol in saliva is detected and measured as a target substance to be detected.
  • the photonic crystal 65 a sheet of a cycloolefin polymer having a predetermined fine structure formed on the surface by thermal nanoimprinting is cut into a predetermined size.
  • FIG. 46 is a flowchart showing an example of the target substance detection method according to the third embodiment.
  • a cortisol antibody solution cortisol antibody concentration of 1 ⁇ g / ml to 1000 ⁇ g / ml
  • the reflective surface 69 of the metal film-coated photonic crystal 21 is exposed to the cortisol antibody solution for a predetermined time at a predetermined temperature or a predetermined temperature if necessary.
  • the cortisol antibody is adsorbed on the reflection surface 69 of the metal film-coated photonic crystal 21.
  • step S102 a phosphate buffer solution (PBS: Phosphate buffered saline) is brought into contact with the reflective surface 69 of the metal film-coated photonic crystal 21. Thereafter, a rinsing process is performed a plurality of times for removal by centrifugal force or the like.
  • PBS Phosphate buffered saline
  • step S103 skim milk is brought into contact with the reflecting surface 69 of the metal film-coated photonic crystal 21 as the blocking agent 75.
  • the reflective surface 69 of the metal film-coated photonic crystal 21 is exposed to skim milk for a predetermined time at a predetermined time or a predetermined temperature if necessary. In this manner, skim milk is adsorbed to the non-adsorbing portion of the cortisol antibody on the reflection surface 69 of the metal film-coated photonic crystal 21.
  • step S104 the rinsing process is performed a plurality of times with the phosphate buffer as in the rinsing process (step S102).
  • a predetermined treatment is performed on the reflection surface 69 of the metal film-coated photonic crystal 21 to form the photonic crystal biosensor 11c.
  • step S105 the light detection unit 12 detects the reflected light LR from the reflection surface 69 when the reflection surface 69 of the photonic crystal 65 is irradiated with light, and the control unit 13c measures the reflected light LR.
  • the control unit 13c measures the spectrum of the reflected light intensity of the reflected light LR.
  • the wavelength of the light (incident light LI) applied to the reflecting surface 69 is, for example, not less than 300 nm and not more than 2000 nm.
  • saliva is first prepared as a solution containing cortisol.
  • Pretreatment such as saliva sampling and impurity removal is performed using, for example, a commercially available saliva collection kit.
  • the preparation of saliva may be performed at any time before the saliva is brought into contact with the photonic crystal biosensor 11c. For example, it may be performed before the photonic crystal biosensor 11c is formed, may be performed in parallel with the formation of the photonic crystal biosensor 11c, or may be performed after the reflected light intensity is measured. 10 ⁇ L to 50 ⁇ L of saliva after sampling and pretreatment is brought into contact with the photonic crystal biosensor 11c.
  • step S107 the reflective surface 69 of the metal film-covered photonic crystal 21 is exposed to a solution containing cortisol for a predetermined time at a predetermined time, if necessary, at a predetermined temperature. In this way, an antigen-antibody reaction is performed.
  • the antigen-antibody reaction in step 17 is performed when the solution is circulated in step S2 in FIG.
  • step S108 the rinsing process is performed a plurality of times with the phosphate buffer solution in the same manner as the rinsing process (step S104).
  • step S109 the target substance detection device 10c is used to irradiate the reflective surface 69 of the metal film-coated photonic crystal 21 with light.
  • the light irradiated at this time is the same as the light irradiated on the reflecting surface 69 in step S15.
  • the target substance detection device 10c measures the reflected light LR from the reflecting surface 69, for example, the spectrum of the reflected light intensity.
  • the wavelength at the extreme value of the reflected light intensity of the photonic crystal biosensor 11c changes under the influence of an antigen-antibody reaction or the like in the vicinity of the reflecting surface 69 or the reflecting surface 69. For this reason, cortisol in saliva can be detected from the difference in wavelength at the extreme value of reflected light intensity before and after the reaction, that is, the amount of wavelength shift. Further, the concentration of cortisol in saliva can be obtained from the wavelength shift amount.
  • the control unit 13c obtains the wavelength shift (wavelength shift amount) at the extreme value (minimum value) of the reflected light intensity (or reflectance) measured in step S109.
  • the wavelength shift amount is, for example, the extreme value (minimum value) of the wavelength ⁇ 2 after the target material is captured on the reflective surface 69 and the reflected light intensity (or reflectance) when the target material is not captured on the reflective surface 69. ) Is the difference ⁇ 2 ⁇ 1 with respect to the wavelength ⁇ 1.
  • step S111 the control unit 13c determines that cortisol is present in saliva, for example, when there is a wavelength shift amount equal to or greater than a predetermined amount. Moreover, the control part 13c determines the density
  • the wavelength shift amount is obtained using the extreme wavelength of the reflected light intensity on the reflecting surface 69 in a state where the target substance is not captured, but the present invention is not limited to this.
  • Step S15 and Step S19 when there are a plurality of extreme values, the extreme value to be focused is appropriately selected. Then, the wavelength ⁇ 1 and the wavelength ⁇ 2 are obtained for the selected extreme value.
  • the metal film-covered photonic crystal 21 has the antibody 74 immobilized on the reflection surface 69, but the present invention is not limited to this, and the metal film-covered photonic crystal 21 has the reflection surface 69. Alternatively, the antibody 74 may be used without being immobilized.
  • the target substance capturing apparatus according to the fourth embodiment is different from that in which the antigen (target substance) 76 is fixed to the reflecting surface 69 of the metal film-coated photonic crystal 21 and the antibody 74 is adsorbed on the antigen 76. Since this is the same as that of the third embodiment, a duplicate description is omitted.
  • 47 to 51 are diagrams for explaining the principle of the photonic crystal biosensor.
  • a specific reaction between the antibody 74 and the antigen 76 will be described using cortisol as the antigen 76 and an anti-cortisol antibody as the antibody 74.
  • the photonic crystal biosensor 11c is similar to the means for fixing the antibody 74 to the reflection surface 69 as the means for fixing the antigen 76 to the reflection surface 69 of the metal film-coated photonic crystal 21. It can be carried out.
  • means for fixing the antigen 76 to the reflecting surface 69 include chemical bonding and physical bonding methods such as covalent bonding, chemical adsorption, and physical adsorption. These means can be appropriately selected according to the properties of the antigen 76.
  • the amount of the antigen 76 fixed to the metal film-coated photonic crystal 21 is a fixed amount. Thereby, when the antibody 74 is adsorbed to the antigen 76 fixed to the metal film-coated photonic crystal 21 to form the complex 65 (see FIGS. 49 and 50), the amount of the complex 77 formed and The correlated physical quantity can be output by the photonic crystal biosensor 11c.
  • the fixed amount of the antigen 76 to be fixed may be changed as appropriate, and can be set to an optimum amount depending on the range of the amount of the antigen 76 contained in the sample S, for example.
  • the blocking agent 75 is fixed to the portion of the reflecting surface 69 where the antigen 76 is not attached.
  • the reflective surface 69 of the photonic crystal 65 is irradiated with light (incident light) LI of, for example, 300 nm or more and 900 nm or less as parallel light so that the optical axis is orthogonal to the reflective surface 69.
  • a wavelength at which the intensity or reflectance of the reflected light LR at this time becomes an extreme value (minimum value in this example) is ⁇ 1.
  • a complex 77 containing a complex 77 of an antigen 76 and an antibody 74 and an antibody 74 is prepared.
  • the mixture M is obtained by mixing the sample S containing the antigen 76 and the solution G containing a known amount of the antibody 74.
  • the complex 77 is obtained by reacting the antibody 74 and the antigen 76 by mixing the sample S containing the antigen 76 and the solution G containing a known amount of the antibody 74.
  • the concentration of the solution G is adjusted in advance so that the total amount of binding sites with the antigen 76 of the antibody 74 contained in the solution G is larger than the total amount of the antigen 76 contained in the sample S.
  • the mixture M is brought into contact with the reflection surface 69 of the metal film-coated photonic crystal 21.
  • a complex 77 is formed on the reflecting surface 69 by the antigen 76 and the antibody 74 fixed on the reflecting surface 69.
  • light (incident light) LI of, for example, 300 nm or more and 2000 nm or less is parallel light and the optical axis is orthogonal to the reflecting surface 69 on the reflecting surface 69 of the metal film-covered photonic crystal 21. Irradiate.
  • the wavelength at which the reflected light intensity or reflectance of the reflected light LR becomes an extreme value is ⁇ 2.
  • the wavelength shift amount of the wavelength at which the light reflectance is an extreme value is ⁇ 2 ⁇ 1.
  • the amount of wavelength shift changes according to the change in the surface state of the reflection surface 69 of the metal film-coated photonic crystal 21.
  • the antigen 76 is detected and quantified.
  • the photonic crystal biosensor 11c outputs an optical physical quantity. This physical quantity correlates with a change in the surface state on the reflecting surface 69 and correlates with the amount of the complex 77 formed by the antigen 76 and the antibody 74 immobilized on the reflecting surface 69.
  • cortisol which is an antigen 76
  • an anti-cortisol antibody which is an antibody 74
  • the metal as in the fourth embodiment.
  • X be the amount of the site to which antigen 76 is bound in sample S
  • C be the known amount of antibody 74 in mixture M.
  • the relationship between X and C is such that X is less than C (X ⁇ C).
  • the antigen 76 reacts with the antibody 74 to form a complex 77. Since X is less than C (X ⁇ C), the amount of antibody 74 in mixture M is CX.
  • the antibody 74 in the mixture M undergoes an antigen-antibody reaction with the antigen 76 on the reflective surface 69 to form a complex 77.
  • the amount of the antigen 76 fixed to the reflecting surface 69 is equal to or greater than the amount CX of the antibody 74 in the mixture M.
  • the amount of the complex 77 becomes CX.
  • the relationship between the amount of the composite 77 fixed to the reflecting surface 69 and the wavelength shift amount ⁇ is obtained in advance. From the above relational expression, the amount X of the antigen 76 can be obtained by C ⁇ / k.
  • the concentration of the antigen 76 can be determined based on the amount X of the antigen 76.
  • the photonic crystal biosensor 11c fixes, for example, a secondary antibody that specifically reacts with the complex 65 to the reflection surface 69 of the metal film-coated photonic crystal 21 as a complex binding substance. You may make it react with the composite_body
  • the secondary antibody is brought into contact with the reflecting surface 69 of the metal film-coated photonic crystal 21 in an excess amount than the first complex (complex) 77. Then, a secondary antibody is added to all the complexes 77 to form a second complex. By doing so, the change in the surface state of the metal film-coated photonic crystal 21 is further increased. As a result, the sensitivity of the photonic crystal biosensor 11c further increases.
  • the secondary antibody may be used as it is, or may be used after adding other substances. Since the change in the surface state of the metal film-coated photonic crystal 21 increases as the secondary antibody increases, the photonic crystal biosensor 11c is reacted with the complex 77 after adding another substance to the secondary antibody. The sensitivity is further increased.
  • the reflective surface 69 after the formation of the second composite is irradiated with light.
  • the wavelength at which the reflected light intensity or reflectance obtained as a result is an extreme value (minimum value in this example) is ⁇ 2.
  • the extreme value of interest is appropriately selected.
  • the wavelength ⁇ 1 and the wavelength ⁇ 2 are obtained for the selected arbitrary extreme value.
  • the photonic crystal biosensor 11c outputs an optical physical quantity. This physical quantity correlates with a change in the surface state of the reflecting surface 69 and correlates with the amount of the second complex fixed to the reflecting surface 69. Thereby, the second complex is detected and quantified. Since the amount of the second complex is the same as the amount of the complex 77, the complex 77 can be quantified.
  • the constituent elements of the first embodiment, the second embodiment, and the third embodiment described above include those that can be easily assumed by those skilled in the art, those that are substantially the same, and those in a so-called equivalent range. Furthermore, the above-described components can be appropriately combined. In addition, various omissions, substitutions, and changes of the components can be made without departing from the scope of the present embodiment.
  • Target Substance Detection Device 11 Photonic Crystal Biosensor (Target Substance Capture Device) 12 light detection unit 13 processing unit 13c control unit LI incident light LR reflected light

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Abstract

L'invention concerne un dispositif de capture de substance cible (10) qui comporte : un élément de support qui supporte une structure revêtue d'une couche métallique qui capture une substance cible et qui est montée sur l'élément de support, et qui comporte au moins deux trous qui s'ouvrent au niveau d'une partie de l'élément de support autre que la partie sur laquelle la structure revêtue d'une couche métallique est montée ; un élément de retenue qui est utilisé avec l'élément de support pour prendre en sandwich la structure revêtue d'une couche métallique entre eux et qui comporte une ouverture au niveau de laquelle les trous de l'élément de support chevauchent la partie capturant la substance cible de la structure revêtue d'une couche métallique qui est montée sur l'élément de support ; et un élément de couverture qui est translucide et qui recouvre l'ouverture de l'élément de retenue.
PCT/JP2014/061899 2013-04-30 2014-04-28 Dispositif de capture de substance cible et dispositif de détection de substance cible Ceased WO2014178385A1 (fr)

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JP2013-095974 2013-04-30
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JP2013095947A JP2014215289A (ja) 2013-04-30 2013-04-30 標的物質捕捉装置
JP2013-095947 2013-04-30
JP2013095953 2013-04-30
JP2013095974A JP2014215291A (ja) 2013-04-30 2013-04-30 標的物質捕捉装置及び標的物質検出装置
JP2014025826A JP2014232098A (ja) 2013-04-30 2014-02-13 標的物質捕捉装置及び標的物質検出装置
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TWI864743B (zh) * 2023-05-09 2024-12-01 友達光電股份有限公司 液體監測觀察容器

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JPH11326193A (ja) * 1998-05-19 1999-11-26 Hitachi Ltd センサおよびこれを利用した測定装置
JP2007502435A (ja) * 2003-05-30 2007-02-08 アプレラ コーポレイション ハイブリダイゼーションおよびｓｐｒ検出のための装置および方法
JP2006242912A (ja) * 2005-03-07 2006-09-14 Fuji Photo Film Co Ltd 送液装置及び送液方法
JP2007192806A (ja) * 2005-12-22 2007-08-02 Canon Inc 標的物質検出素子用基板、標的物質検出素子、それを用いた標的物質の検出装置及び検出方法、並びにそのためのキット
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TWI864743B (zh) * 2023-05-09 2024-12-01 友達光電股份有限公司 液體監測觀察容器

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