WO2013161991A1 - Capteur de fluorescence - Google Patents

Capteur de fluorescence Download PDF

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Publication number
WO2013161991A1
WO2013161991A1 PCT/JP2013/062368 JP2013062368W WO2013161991A1 WO 2013161991 A1 WO2013161991 A1 WO 2013161991A1 JP 2013062368 W JP2013062368 W JP 2013062368W WO 2013161991 A1 WO2013161991 A1 WO 2013161991A1
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WO
WIPO (PCT)
Prior art keywords
fluorescence
indicator
sensor
analyte
light
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PCT/JP2013/062368
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English (en)
Japanese (ja)
Inventor
亮 太田
Original Assignee
オリンパス株式会社
テルモ株式会社
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Filing date
Publication date
Application filed by オリンパス株式会社, テルモ株式会社 filed Critical オリンパス株式会社
Publication of WO2013161991A1 publication Critical patent/WO2013161991A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14503—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue invasive, e.g. introduced into the body by a catheter or needle or using implanted sensors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14532—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue for measuring glucose, e.g. by tissue impedance measurement
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2562/00—Details of sensors; Constructional details of sensor housings or probes; Accessories for sensors
    • A61B2562/12—Manufacturing methods specially adapted for producing sensors for in-vivo measurements
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G01N21/80—Indicating pH value

Definitions

  • the present invention relates to a fluorescent sensor that measures the concentration of an analyte, and more particularly to a fluorescent sensor that is a micro-fluorescence spectrophotometer manufactured using semiconductor manufacturing technology and MEMS technology.
  • Various analyzers have been developed to confirm the presence of analytes in liquids, that is, substances to be measured, or to measure concentrations.
  • a fluorescent dye whose properties change due to the presence of the analyte and generates fluorescence and a solution to be measured containing the analyte are injected, and excitation light is irradiated to increase the fluorescence intensity from the fluorescent dye.
  • Fluorescence spectrophotometers that measure analyte concentration by measuring are known.
  • a small fluorescent spectrophotometer has a photodetector and an indicator containing a fluorescent dye. And, by irradiating the indicator into which the analyte in the solution to be measured can enter the excitation light from the light source, the fluorescent dye in the indicator generates a fluorescence having a light amount corresponding to the analyte concentration in the solution to be measured, The fluorescence is received by the photodetector.
  • the photodetector is a photoelectric conversion element and outputs an electrical signal corresponding to the amount of received light. The analyte concentration in the solution to be measured is measured from this electrical signal.
  • microfluorescence spectrophotometer manufactured using semiconductor manufacturing technology and MEMS technology.
  • the microfluorometer is referred to as a “fluorescence sensor”.
  • a fluorescent sensor 110 shown in FIGS. 1 and 2 is disclosed in US Pat. No. 5,039,490.
  • the fluorescence sensor 110 includes an optical plate-like portion 105 having a transparent support substrate 101 that can transmit excitation light, a photoelectric conversion element 103 that converts fluorescence into an electrical signal, and a condensing function portion 105A that collects excitation light.
  • the indicator 117 is configured to generate fluorescence by interacting with the analyte 9 by the excitation light, and the cover layer 109.
  • the photoelectric conversion element 103 is formed on a substrate 103A made of, for example, silicon.
  • the substrate 103A does not transmit excitation light.
  • the fluorescence sensor 110 has a void region 120 that can transmit excitation light around the photoelectric conversion element 103.
  • Fluorescence F is generated by the interaction between the condensed excitation light E and the analyte 9 that has entered the indicator 117. Part of the generated fluorescence enters the photoelectric conversion element 103, and a signal such as a current or voltage proportional to the fluorescence intensity, that is, the concentration of the analyte 9, is generated in the photoelectric conversion element 103.
  • the excitation light does not enter the photoelectric conversion element 103 due to the action of a filter (not shown) formed on the photoelectric conversion element 103.
  • the photodiode which is the photoelectric conversion element 103 is formed on the substrate 103A laminated on the transparent support substrate 101, and the optical plate portion 105 and the indicator 117 are formed thereon. Are stacked.
  • the above-described fluorescent sensor 110 has the gap region 120 which is a passage of excitation light and the region of the photoelectric conversion element 103 on the same plane. For this reason, if the area of the gap region 120, which is a passage, is increased in order to guide more excitation light to the indicator 117, the area of the photoelectric conversion element 103 is reduced, and thus the sensitivity of the fluorescent sensor is not increased. . On the other hand, if the area of the photoelectric conversion element 103 is increased in order to increase the detection sensitivity of the photoelectric conversion element 103, the area of the gap region 120, which is the path of the excitation light, is reduced, and the excitation light guided to the indicator 117 is reduced. Therefore, the sensitivity of the fluorescence sensor is not increased. That is, with the fluorescent sensor 110, it is not easy to obtain high detection sensitivity.
  • An object of the present invention is to provide a fluorescent sensor with high detection sensitivity.
  • the fluorescent sensor of one embodiment of the present invention includes a substrate portion on which a plurality of recesses are formed on a main surface, and a photoelectric conversion element that receives fluorescence and outputs a detection signal is formed on a wall surface of each recess, A filter that covers the photoelectric conversion element and blocks excitation light; an indicator that is disposed inside each of the recesses; and that generates the fluorescence corresponding to the amount of analyte when receiving the excitation light; and The light-shielding layer that covers the opening and blocks external light from entering the indicator but through which the analyte passes and a light-emitting element that irradiates the indicator with excitation light from the bottom surface side of the recess.
  • the fluorescence sensor 4 constitutes a sensor system 1 together with the main body 2 and the receiver 3. That is, the sensor system 1 includes a fluorescent sensor 4, a main body 2, and a receiver 3 that receives and stores a signal from the main body 2. Transmission / reception of signals between the main body 2 and the receiver 3 is performed wirelessly or by wire.
  • the needle-type fluorescent sensor 4 includes a needle portion 7 having a needle tip portion 5 having a sensor portion 10 as a main functional portion and an elongated needle body portion 6, and a connector portion integrated with a rear end portion of the needle body portion 6. 8 and. Needle tip 5, needle body 6 and connector 8 may be integrally formed of the same material.
  • the connector part 8 is detachably fitted to the fitting part 2A of the main body part 2.
  • the plurality of wirings 60 extending from the sensor unit 10 of the fluorescent sensor 4 are electrically connected to the main body unit 2 when the connector unit 8 is mechanically fitted to the fitting unit 2A of the main body unit 2. .
  • the main body unit 2 includes a control unit 2B including a CPU that drives and controls the sensor unit 10, and a calculation unit 2C including a CPU that processes a signal output from the sensor unit 10.
  • a control unit 2B including a CPU that drives and controls the sensor unit 10 and a calculation unit 2C including a CPU that processes a signal output from the sensor unit 10.
  • the control unit 2B and the calculation unit 2C may be disposed in the connector unit 8 of the fluorescent sensor 4 or may be disposed in the receiver 3. Further, the control unit 2B and the calculation unit 2C may be the same CPU.
  • the main body 2 when performing wired transmission / reception with the receiver 3, the main body 2 has a signal line instead of a wireless antenna. Further, when the main body 2 has a memory unit having a necessary capacity, the receiver 3 is not necessary.
  • the fluorescence sensor 4 is a needle-type sensor that measures an analyte in the body, and is a short-term subcutaneous indwelling type with a continuous use period of about one week.
  • the collected bodily fluid or the bodily fluid that circulates inside the body via a flow path outside the body may be contacted outside the body without inserting the fluorescent sensor 4 into the body.
  • the sensor unit 10 which is the main functional unit of the fluorescence sensor 4 includes a substrate unit 20 and a light emitting diode (Light Emitting Diode: hereinafter referred to as “LED”) that generates excitation light. 15A and 15B, indicators 17A and 17B that generate fluorescence corresponding to the excitation light and the amount of analyte, and a light shielding layer 18.
  • LED Light Emitting Diode
  • 15A and 15B indicators 17A and 17B that generate fluorescence corresponding to the excitation light and the amount of analyte
  • a light shielding layer 18 In the fluorescent sensor 4, the analyte 9 passes through the light shielding layer 18 and enters the indicator 17 when the light shielding layer 18 comes into contact with blood or body fluid in the living body.
  • the substrate unit 20 includes a wiring substrate unit 30 that is a first substrate unit and a frame-shaped substrate unit that is a second substrate unit in which two through holes 46A and 46B are formed on the main surface 20SA (main surface 20SB). 40 are bonded to each other through an adhesive layer 13.
  • substrate part 20 is a multicavity with two recessed part 23A, 23B in main surface 20SA. That is, the surface of the wiring board portion 30 is the bottom surface of the recesses 23A and 23B, and the wall surfaces of the through holes 46A and 46B of the frame-like substrate portion 40 are the wall surfaces of the recesses 23A and 23B.
  • an insulating layer is appropriately formed on the surface of the substrate portion 20 made of, for example, an N-type semiconductor, that is, the wiring substrate portion 30 and the frame-like substrate portion 40, but is not shown. Further, hereinafter, when referring to each component having the same function, the last letter (A or B) is omitted.
  • each of the through holes 46A and 46B is referred to as a through hole 46.
  • the indicator 17A and the LED 15A are disposed inside the recess 23A, and the indicator 17B and the LED 15B are disposed inside the recess 23B.
  • the light emitting element is not limited to the LED. However, a substantially rectangular chip-shaped LED is preferable from the viewpoints of light generation efficiency, wide wavelength selectivity of excitation light, and generation of only light having a wavelength other than ultraviolet light serving as excitation light.
  • the indicator 17 generates fluorescence with a light amount corresponding to the amount of the analyte 9 by the interaction with the analyte 9 that has entered and the excitation light.
  • the indicator 17 generates fluorescence having a longer wavelength, for example, a wavelength of 460 nm with respect to excitation light having a wavelength of 375 nm.
  • the thickness of the indicator 17 is set to about several tens ⁇ m to 200 ⁇ m.
  • the indicator 17 is composed of a base material containing a fluorescent dye that generates fluorescence having an intensity corresponding to the amount of the analyte 9, that is, the concentration of the analyte in the sample.
  • the fluorescent dye is selected according to the type of the analyte 9 and can be used for any fluorescent dye in which the amount of fluorescence generated according to the amount of the analyte 9 changes reversibly. That is, the fluorescent sensor 4 corresponds to various uses such as an oxygen sensor, a glucose sensor, a pH sensor, an immunosensor, or a microorganism sensor, depending on the selection of the fluorescent dye.
  • the indicator 17 includes, for example, a hydrogel that easily contains water as a base material and contains or is bonded to the fluorescent dye in the hydrogel.
  • Hydrogel components are prepared from acrylic hydrogels prepared by polymerizing polysaccharides such as methylcellulose or dextran, monomers such as (meth) acrylamide, methylolacrylamide, or hydroxyethyl acrylate, or from polyethylene glycol and diisocyanate. Urethane hydrogel can be used.
  • the indicator 17 may be joined to the LED 15 through a transparent protective layer that protects the LED 15.
  • a transparent protective layer silicon oxide, epoxy resin, silicone resin, or transparent amorphous fluororesin can be used.
  • the transparent protective layer is selected from materials having characteristics such as electrical insulation, moisture barrier properties, and good transmittance for excitation light and fluorescence. As a characteristic of the transparent protective layer, it is important that the generation of fluorescence in the layer is small even when irradiated with excitation light. Needless to say, the characteristic that the fluorescence is small is an important characteristic of all the transparent materials of the fluorescent sensor 4 except the indicator.
  • the light shielding layer 18 is a layer formed on the upper surface side of the indicator 17 and having a thickness of several tens of ⁇ m or less.
  • the light shielding layer 18 may be divided for the indicator 17A and the indicator 17B.
  • a photodiode (Photo Diode: hereinafter referred to as “PD”) that receives fluorescence and outputs a detection signal.
  • the element 12A is also formed, and the PD element 12B is also formed on the wall surface of the through hole 46B. That is, the light receiving portion 12 ⁇ / b> D of the PD element 12 is provided so as to surround the indicator 17, and is formed so that the light receiving surface faces the indicator 17.
  • the light receiving unit 12D is referred to as a PD element 12.
  • the PD element 12 may be formed on the entire wall surface, but may be formed only in a region facing the indicator 17 in order to efficiently receive only fluorescence.
  • the PD element 12 may be formed on all four wall surfaces, or may be formed on only a part of the surfaces.
  • the PD element 12 may be formed on at least a part of the wall surface of the recess 23.
  • the photoelectric conversion element may be a photoconductor (photoconductor), a phototransistor (Phototransistor, PT), or the like.
  • Filters 14A and 14B are disposed so as to cover the PD elements 12A and 12B formed on the wall surface.
  • the filter 14 covering at least a part of the PD elements 12A and 12B is a high-pass absorption filter that blocks excitation light but allows fluorescence having a wavelength longer than that of the excitation light to pass.
  • a silicon layer or a silicon carbide layer is suitable as a material for such a filter.
  • the transmittance of the silicon layer and the silicon carbide layer is 10 ⁇ 5 % or less at the excitation light wavelength of 375 nm, whereas the transmittance is 10% or more at the fluorescence wavelength of 460 nm (the transmittance of the excitation light wavelength).
  • the filter 14 may be a bandpass filter having a high fluorescence wavelength transmittance.
  • the filter 14 may be arrange
  • the wiring board portion 30 is a wiring layer comprising three wirings 51A, 52, 51B for transmitting detection signals from the PD elements 12A, 12B, and wirings 53, 54 for transmitting drive signals to the LEDs 15A, 15B. 50.
  • the wiring 52 is connected by partially introducing an N-type impurity such as phosphorus or arsenic into the surface of the frame-shaped substrate portion 40, which is an N-type semiconductor, to form a low-resistance region 12H with higher conductivity. Yes.
  • each wiring is arrange
  • the wiring 51 and the wiring 51 ⁇ / b> B may be connected to any one of the plurality of wirings 60 that transmit signals to the main body 2.
  • the material of the frame-shaped substrate portion 40 is preferably single crystal silicon in order to form the PD element 12 on the frame-shaped substrate portion 40, but may be glass or ceramic.
  • a semiconductor thin film such as polysilicon or amorphous silicon of several microns or less is formed on the surface of the recess 23 and the surface of one main surface 20SB of the frame-shaped substrate portion 40, and the PD element 12 is formed thereon.
  • the excitation light generated by the LED 15 is applied to the fluorescent dye in the indicator 17.
  • a part of the fluorescence generated by the interaction of the fluorescent dye with the analyte 9 passes through the filter 14 and reaches the PD element 12 and is converted into a detection signal.
  • the fluorescence sensor 4 has a high detection sensitivity because the PD element 12 formed on the wall surface surrounding the indicator 17 detects fluorescence.
  • the fluorescence sensor 4 Furthermore, in the fluorescence sensor 4, PD elements 12A and 12B are formed on the wall surfaces of the two recesses 23A and 23B, respectively. For this reason, the wall surface area, that is, the area of the light receiving portion 12D of the PD element 12 is large. Further, since the distance from the center of the recess 23 to the light receiving portion of the PD element 12 is short, the fluorescence generated by the fluorescent dye reaches the PD element 12 without being attenuated. For this reason, the fluorescence sensor 4 has a high signal output and a high dynamic sensitivity with a good S / N ratio, and therefore has high detection sensitivity.
  • 6A to 6E are partial cross-sectional views of the region of one fluorescent sensor 4, but in the actual process, a large number of sensor portions 10 are collectively formed as a wafer process.
  • the conductive (N-type) silicon wafer 40W is etched through the mask layer 71 to obtain a large number of through holes 46A, 46B is formed.
  • Various known methods can be used for etching.
  • the size of the opening of the through hole 46 is designed according to the specifications. However, since the location is the needle tip portion 5, the plurality of openings have an elongated shape such as 150 ⁇ m in length and 500 ⁇ m in width, for example.
  • the one opening is preferably about 10 ⁇ m to several hundred ⁇ m.
  • the wall surface of the through hole 46 is perpendicular to the main surface, but the wall surface may have a predetermined angle, that is, a tapered shape, as will be described later.
  • the tapered recess can be produced, for example, by wet etching.
  • the PD element 12 is formed on the wall surface of the through hole 46. That is, the ion implantation process is performed from four directions in a state where the silicon wafer 40W on which the mask layer 72 is formed is inclined by 5 degrees to 30 degrees.
  • the conditions for implanting boron (B) are acceleration voltage: 10 to 100 keV and implantation amount: about 1 ⁇ 10 15 cm ⁇ 2 .
  • the filter 14 is formed by the CVD method on the PD element 12 on the wall surface of the through hole 46 of the silicon wafer 40W.
  • a conductive (N-type) silicon wafer 30 ⁇ / b> W to be the wiring board unit 30 is prepared.
  • an insulating film such as an oxide film is formed on the silicon wafer 30W, drive signals are sent to the wirings 51, 52, 51B and the LEDs 15A, 15B for transmitting the detection signals from the PD element 12.
  • Wirings 53 and 54 for supply are formed by sputtering or vapor deposition.
  • the silicon wafer 40W is turned upside down, and the main surface 20SB of the silicon wafer 40W is bonded to the silicon wafer 30W through the adhesive layer 13.
  • the through hole 46 of the frame-shaped substrate portion 40 becomes a recess 23 having a bottom surface.
  • LEDs 15A and 15B are disposed on the bottom surfaces of the recesses 23A and 23B of the bonded wafer 20W.
  • a bonding method using an optically transparent acrylic resin or silicone resin, or various bonding methods such as a flip chip bonding method can be used.
  • the indicator 17A, 17B is arrange
  • the light shielding layer 18 is formed on the indicators 17A and 17B, and the sensor substrate 10W is completed.
  • the light shielding layer 18 has a submicron pore structure, an inorganic thin film such as metal or ceramic, or a composite structure with hydrogels in which carbon black is mixed in a base material of organic polymer such as polyimide or polyurethane, or Further, a resin obtained by mixing carbon black into an analyte-permeable polymer such as celluloses or polyacrylamide, or a resin obtained by laminating them is used.
  • a large number of sensor units 10 are manufactured at once by separating the sensor substrate 10W into individual pieces. And the fluorescence sensor 4 is completed by joining the sensor part 10 with the front-end
  • the manufacturing method of the fluorescent sensor is not limited to this, and the LED 15 and the like are arranged in the recess 23 after joining the separated wiring board part 30 and the separated frame-like board part 40.
  • a method such as setting may be used.
  • the first silicon wafer may be processed so that the extended portion of the wiring board portion 30 constitutes the needle main body portion 6 of the needle portion 7, or the separately prepared needle main body portion 6 and the fluorescence sensor 4 are provided. You may comprise the needle part 7 by joining the needle front-end
  • the fluorescent sensor 4 can be mass-produced by a wafer process. For this reason, the fluorescence sensor 4 can provide stable quality at low cost.
  • the LED 15 emits pulsed excitation light having a center wavelength of around 375 nm at intervals of, for example, once every 30 seconds.
  • the pulse current to the LED 15 is 1 mA to 100 mA
  • the pulse width of light emission is 101 ms to 100 ms.
  • the excitation light generated by the LED 15 enters the indicator 17.
  • the indicator 17 emits fluorescence having an intensity corresponding to the amount of the analyte 9.
  • the analyte 9 passes through the light shielding layer 18 and enters and exits the indicator 17.
  • a part of the fluorescence generated by the indicator 17 enters the PD element 12 through the filter 14. Fluorescence is photoelectrically converted in the PD element 12 to generate photogenerated charges, which are output as detection signals. Part of the excitation light generated by the LED 15 is incident on the wall surface of the recess 23, but hardly enters the PD element 12 due to the action of the filter 14.
  • the calculation unit 2C of the main body 2 performs calculation processing based on the detection signal, that is, the current caused by the photogenerated charge from the PD element 12 or the voltage caused by the accumulated photogenerated charge. Calculate the amount of light.
  • the fluorescence sensor 4 has two sets of sensor function units capable of independently measuring the analyte, that is, two sets of the LED 15 and the PD element 12. For this reason, the fluorescence sensor 4 can exhibit a specific effect by various driving methods.
  • the LED 15A and the LED 15B may be caused to emit light alternately.
  • the signal output decreases, the fluorescent sensor 4 can perform measurement for a longer time (longer life).
  • the measurement can be continued if the other is operating normally, and thus the fluorescence sensor 4 is improved in reliability. Further, usually, measurement is performed using one sensor function unit, and the other may be used for backup.
  • the indicator 17A and the indicator 17B may be in different deterioration states by changing the integrated light amount irradiated to the LED 15A and the integrated light amount irradiated to the LED 15B. Since it is possible to correct a decrease in the amount of fluorescent light due to deterioration, the fluorescent sensor 4 becomes more accurate.
  • the fluorescent sensor 4 having two sets of sensor function units has effects such as high output, high reliability, long life, and high accuracy by selecting a control method according to the purpose.
  • the fluorescence sensor 4A of the second embodiment will be described. Since the fluorescence sensor 4A is similar to the fluorescence sensor 4 of the first embodiment, the same components are denoted by the same reference numerals and description thereof is omitted.
  • the sensor unit 10A includes a frame-shaped substrate unit 40A and a wiring substrate unit 30A on which one LED 15L is mounted to join the substrate unit 20A. It is composed. That is, one LED 15L irradiates excitation light from below to the two indicators 17A and 17B filled in the two recesses 23A and 23B.
  • the PD element 12A and the PD element 12B are connected, and a detection signal is output via the common wirings 51 and 52.
  • the fluorescent sensor 4A has a simple structure because one LED 15L is used as a common excitation light source for the two indicators 17A and 17B.
  • the fluorescence sensor 4B of the third embodiment will be described. Since the fluorescence sensor 4B is similar to the fluorescence sensors 4 and 4A, the same components are denoted by the same reference numerals and description thereof is omitted.
  • the substrate portion 20 ⁇ / b> B of the sensor portion 10 ⁇ / b> B is a region directly below the opening on the side opposite to the opening covered with the light shielding layer 18.
  • One LED 15L is joined to the frame-shaped substrate portion 40B so as to cover the frame.
  • the LED 15L covers the region immediately below the opening on the main surface 20SB side of the two through holes 46A and 46B.
  • the bottom surfaces of the through holes 46A and 46B are constituted by the upper surface of the LED 15L.
  • the bottom surfaces of the through holes 46A and 46B may be the transparent adhesive layer 13.
  • the wiring layer 50 which comprises the wiring connected with PD element 12A, 12B and LED15L is arrange
  • the light leakage prevention layer 19 disposed so as to cover the bottom surface (lower surface) and the side surface of the LED 15L is excited by the excitation light emitted from the bottom surface and the side surface and the excitation reflected by the second main surface 20SB of the frame-shaped substrate portion 40B. Prevent light from leaking outside. That is, the light leakage prevention layer 19 has a function similar to that of the light shielding layer 18, but does not require analyte permeability.
  • the fluorescence sensor 4B has the effects of the fluorescence sensors 4 and 4A, and further has a simple configuration, so that it is easier to manufacture.
  • the substrate portion 20C constituting the sensor portion 10C is integrally made of silicon which is a semiconductor. That is, the recesses 23A and 23B of the substrate portion 20C are recesses formed by an etching method.
  • etching method a wet etching method using a tetramethylammonium hydroxide (TMAH) aqueous solution, a potassium hydroxide (KOH) aqueous solution, or the like is preferable, but dry etching such as reactive ion etching (RIE) or chemical dry etching (CDE) is used.
  • TMAH tetramethylammonium hydroxide
  • KOH potassium hydroxide
  • CDE chemical dry etching
  • PD elements 12A and 12B are formed on the wall surfaces of the recesses 23A and 23B, respectively.
  • the concave portion 23 having the wall surface taper not only has a larger area for forming the PD element than the concave portion having a vertical wall surface, but also facilitates the formation of the PD element 12.
  • the PD elements 12A and 13A and the LEDs 15A and 15B are connected to the wiring layer 50 of the second main surface 20SB through the through wiring.
  • the fluorescence sensor 4C has the effects of the fluorescence sensor 4 and is simpler in configuration, and therefore easier to manufacture.
  • the sensor units 10 to 10C have the two concave portions 23A and 23B that are rectangular in a plan view (cross-sectional shape).
  • the number, that is, the number of indicators 17 may be three or more.
  • it may have four recesses 23A to 23D like a fluorescent sensor 4D (sensor unit 10D) of the modification shown in FIG. 10B.
  • the cross-sectional shape of the plurality of recesses 23 may be a polygon such as a hexagon or an ellipse, or various shapes such as a combination thereof. But you can.
  • the fluorescence sensor has high detection sensitivity.
  • the intensity of the excitation light generated by the LED 15 has an in-plane distribution.
  • the fluorescence intensity emitted from the indicator 17 in the region irradiated with the high-intensity excitation light is high.
  • the fluorescence intensity emitted by the indicator 17 in the region where the intensity of the incident excitation light is low is low. That is, if the intensity of the excitation light incident on the indicator 17 has a large in-plane distribution, the detection sensitivity of the fluorescent sensor may be reduced, or deterioration with time may be accelerated.
  • the intensity distribution of the excitation light incident on the indicator 17 can be averaged by the opening area, shape, arrangement, and the like of the plurality of recesses 23.
  • Fluorescence sensors 4D to 4D2 of Modification 1 can not only have higher detection sensitivity, but also suppress the deterioration of indicator 17 in addition to the effects of fluorescence sensors 4 to 4C.
  • the plurality of indicators 17 and the plurality of PD elements 12 measure the same type of analyte 9, for example, glucose. That is, the fluorescence sensors 4 to 4D2 correspond to various applications such as an oxygen sensor, a glucose sensor, a pH sensor, an immunosensor, or a microorganism sensor, depending on the selection of the fluorescent dye. However, only one type of analyte 9 can be measured.
  • the sensor unit 10E of the fluorescence sensor 4E of the modification 2 shown in FIG. 11 is a multi-sensing type capable of measuring a plurality of types of analytes.
  • the indicator generates a first indicator 17A1 that generates fluorescence according to the amount of the first analyte 9A, and a second indicator 17B1 that generates fluorescence according to the amount of the second analyte 9B. And consist of
  • the first indicator 17A that fills the recess 23A includes the phosphor 81A that generates fluorescence of the first wavelength according to the amount of glucose that is the first analyte 9A, and the second indicator that fills the recess 23B.
  • the indicator 17B includes a phosphor 81B that generates fluorescence of the second wavelength corresponding to the hydrogen ion concentration, which is the second analyte 9B.
  • the phosphor 81A includes a phenylboronic acid derivative, and the phosphor 81B includes, for example, CdSe / Zn nanoparticles.
  • both the phosphor 81A and the phosphor 81B receive the excitation light of 375 nm, the phosphor 81A and the phosphor 81B generate fluorescence corresponding to the glucose concentration or the hydrogen ion concentration as the analyte.
  • the computing unit 2C may correct the glucose concentration measured by the first indicator 17A according to the hydrogen ion concentration (pH) measured by the second indicator 17B. Even when the first indicator 17A and the second indicator 17B contain the same phosphor at different concentrations, the concentration of the measured analyte can be corrected.
  • the type of indicator 17 is selected according to the analyte to be measured. Further, a fluorescent sensor having three or more recesses may have a different indicator in each recess.
  • the indicator for example, when measuring potassium ion, a phosphor (fluorescence wavelength: 500 nm) including a crown ether derivative having a fluorescent residue can be used, and when measuring oxygen, Tris (4 , 7-diphenyl-1,10-phenanthroline) a phosphor containing a ruthenium (II) perchlorate molecule (fluorescence wavelength: 530 nm) can be used.
  • the multi-sensing type fluorescence sensor 4E has the effect of the fluorescence sensor 4 and the like, and moreover, since a plurality of analytes can be measured with one sensor, the environment and biological information can be measured more accurately. Furthermore, since the measured analyte concentration can be corrected, the accuracy is higher.
  • the wavelength of the excitation light to be irradiated may be changed depending on the type of analyte to be measured, that is, the type of indicator. That is, the first indicator 17A may be irradiated with the excitation light E1 having the first wavelength, and the second indicator 17B may be irradiated with the excitation light E2 having the second wavelength.
  • the first indicator 17A including a phenylboronic acid derivative as a phosphor is irradiated with a first excitation light E1 having a wavelength of 375 nm
  • the second indicator 17B including a GFP (green fluorescent protein) derivative as a phosphor You may irradiate the 2nd excitation light E2 of wavelength 490nm.
  • the sensor unit 10E of the fluorescence sensor 4E includes an LED 15A1 that is a first light emitting element that generates excitation light E1 of the first wavelength, and excitation light E2 of the second wavelength.
  • LED15B1 which is the 2nd light emitting element which generate
  • the wavelength conversion filter 80 may be used as in the sensor unit 10F of the fluorescence sensor 4F of Modification 3 shown in FIG.
  • the wavelength conversion filter 80 emits the excitation light E1 of the first wavelength of 375 nm generated by the LED 15L to the first indicator 17A as it is, and the excitation light E1 generated by the LED 15L to the second indicator 17B. Is converted into excitation light E2 having a second wavelength of 460 nm and irradiated.
  • the indicators 17A and 17B generate the first phosphor F1 that generates the first fluorescence F1 according to the amount of the first analyte 9A.
  • produces the 2nd fluorescence F2 according to the quantity of the 2nd analyte 9B may be sufficient.
  • the first PD element 12A covered by the first filter 14A1 that selectively transmits the first fluorescence F1 generated by the first phosphor 81A and the second phosphor 81B are generated. If it has 2nd PD element 12B covered with 2nd filter 14B1 which permeate

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Abstract

Le capteur de fluorescence (4) ci-décrit comprend : une partie substrat (20) comportant une pluralité d'évidements (23A, 23B) qui sont formés dans une surface principale (20SA); des éléments PD (12A, 12B) pour recevoir la fluorescence et générer un signal de détection, qui sont formés sur les surfaces de parois des évidements (23A, 23B), respectivement; des filtres (14A, 14B) pour couvrir les éléments PD (12A, 12B) et bloquer la lumière d'excitation; des indicateurs (17A, 17B) pour générer une fluorescence correspondant à une quantité d'analyte à réception de la lumière d'excitation, les indicateurs (17A, 17B) se trouvant à l'intérieur des évidements (23A, 23B), respectivement; une couche faisant écran à la lumière (18) pour couvrir les ouvertures des évidements (23A, 23B) et empêcher la lumière extérieure de pénétrer dans les indicateurs (17A, 17B) mais laissant passer un analyte (9); et des DEL (15A, 15B) pour exposer les indicateurs (17A, 17B) à la lumière d'excitation émanant, respectivement, d'un côté inférieur des évidements (23A, 23B).
PCT/JP2013/062368 2012-04-27 2013-04-26 Capteur de fluorescence WO2013161991A1 (fr)

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JP2012-103605 2012-04-27
JP2012103605 2012-04-27

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11500825A (ja) * 1995-02-21 1999-01-19 アーサー・イー・コルヴィン・ジュニアー 光学式螢光センサ
US20040161853A1 (en) * 2003-02-13 2004-08-19 Zhongping Yang Implantable chemical sensor with rugged optical coupler
WO2010119916A1 (fr) * 2009-04-13 2010-10-21 Olympus Corporation Capteur de fluorescence, capteur de fluorescence de type aiguille et procédé pour mesurer un analyte

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11500825A (ja) * 1995-02-21 1999-01-19 アーサー・イー・コルヴィン・ジュニアー 光学式螢光センサ
US20040161853A1 (en) * 2003-02-13 2004-08-19 Zhongping Yang Implantable chemical sensor with rugged optical coupler
WO2010119916A1 (fr) * 2009-04-13 2010-10-21 Olympus Corporation Capteur de fluorescence, capteur de fluorescence de type aiguille et procédé pour mesurer un analyte

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