JP2005315871A - Intracorporeal substance measuring assembly, manufacturing method for intracorporeal substance measuring assembly, and embedded type substance sensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an intracorporeal substance measuring assembly used for an embedded type substance sensor for measuring an intracorporeal substance, and capable of measuring a measured substance stably for a long time. <P>SOLUTION: This intracorporeal substance measuring assembly provided in the embedded-type substance sensor for detecting and measuring the intercorporeal measured substance has a detection layer 2 containing at least one kind of fluorescent indicator for generating fluorescence in response to the concentration of the measured substance, and an optical separation layer 3 provided on the detection layer 2, optically opaque, allowing the measured substance to permeate therethrough, and preventing at least one out of living body substances having the possibility of deteriorating the detection layer 2 and/or disturbing the fluorescence from being permeated therethrough. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a composition for measuring a substance in a body, a method for manufacturing the composition for measuring a substance in a body, and a method for measuring the substance in the body, provided in an implantable substance sensor for detecting and measuring a substance to be measured in the body. The present invention relates to an implantable material sensor using a composition.

  Implantable sensors are useful for various disease diseases, such as the follow-up of disease states and the monitoring of therapeutic effects, and are one of the fields that have been actively studied in recent years. Particularly in the treatment of diabetes, blood glucose control by continuous blood glucose measurement is said to contribute to the reduction of disease progression and morbidity of complications.

  Many current diabetic patients collect blood samples by puncturing their fingers or the like and supply them to a blood glucose meter to read the measured values for self-management of blood glucose. However, this method has problems in terms of pain and convenience for patients, and it is difficult to measure and grasp the blood glucose level change frequently because the measurement is limited to several times a day. Currently. For these reasons, the usefulness of the implantable continuous blood glucose meter is considered to be high.

  As an in-vivo sensor, for example, a device that inputs and outputs a signal from a device that is implanted in a living body using wireless is disclosed (see Patent Documents 1 and 2). According to this technology, a detection device (sensor) that has an indicator layer that reversibly reacts with glucose and changes its fluorescence characteristics is embedded in a living body, glucose concentration is measured by the change in the amount of fluorescence, and the data is transmitted outside the body as electromagnetic waves. Etc. are derived.

  Moreover, as what can be utilized for the indicator layer of such a detection apparatus, what carried out the covalent bond of the fluorescent substance reversibly couple | bonded with glucose, such as phenyl boronic acid, to the polystyrene is proposed (refer patent document 3).

Moreover, although various sugar components exist in the biological component, a fluorescent substance that can be specifically reversibly bound by glucose has also been proposed (see Patent Document 4).
U.S. Pat. No. 4,550,731 U.S. Pat. No. 4,253,466 US Pat. No. 5,137,833 US Pat. No. 5,503,770

  However, in these conventional implantable sensors, a substance (indicator substance) that emits fluorescence upon contact with a substance to be measured deteriorates during use and cannot withstand long-term implantation, and also in terms of measurement accuracy. There is a problem that accuracy is low.

  Accordingly, an object of the present invention is to provide a composition for measuring a substance in a body that is used in an implantable substance sensor for measuring a substance in the body, and that can stably measure an object to be measured for a long time. It is.

  Another object of the present invention is to provide a method for producing a composition for measuring a substance in a body, which is used in an implantable substance sensor for measuring a substance in the body, and enables a measurement object to be measured stably for a long time. Is to provide.

  Furthermore, an object of the present invention is to provide an implantable substance sensor using an in-vivo substance measuring composition that makes it possible to stably measure an object to be measured for a long time.

  The above objects are achieved by the following means.

  (1) An in-vivo substance measurement component provided in an implantable substance sensor that detects and measures a substance to be measured in a living body, and emits at least one kind of fluorescence according to the concentration of the substance to be measured A composition for measuring an in-vivo substance, comprising: a detection layer containing a fluorescent indicator; and an optical separation layer that is provided on one surface of the detection layer and is optically opaque and transmits a substance to be measured. .

  (2) The detection layer is made of a base material to which the fluorescent indicator is bound.

  (3) When the substance to be measured is a saccharide, the fluorescent indicator is a fluorescent substance containing phenylboronic acid.

  (4) The fluorescent substance containing phenylboronic acid is represented by the chemical formula (1)

  (In the chemical formula (1), Q is a fluorescent residue, and at least one of R1, R2, and R3 is an active group for binding to the substrate of the detection layer; R6 and R7 are at least one substituent selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an allyl group, an allylalkenyl group, a substituted alkyl group, an oxyalkyl group, an acyl group, and a halogen atom) It is what is shown by.

  (5) Q is a fluorescent residue containing anthracene.

  (6) The base material of the detection layer is a polymer substance having optical transparency.

  (7) The optical separation layer is a polymer porous film formed by a phase inversion method.

  (8) The polymer substance is at least one polymer substance selected from the group consisting of cellulose, polyacrylamide, polyethylene glycol, polyvinyl alcohol, and derivatives thereof.

  (9) The optical separation layer is characterized by comprising an opaque substance and a base material carrying the opaque substance.

  (10) The opaque material is characterized by comprising at least one material selected from the group consisting of carbon black, fullerene, and carbon nanotubes.

  (11) The substrate of the optical separation layer is a polymer substance.

  (12) The porous polymer membrane is characterized by comprising at least one of a cellulose derivative, a polysulfone resin, a polyamide resin, and an aromatic polyether ketone resin.

  (13) The polymer substance is a cross-linked dextran.

  (14) The optical separation layer is characterized in that it allows the substance to be measured to pass through and does not allow at least one biological component other than the substance to be measured to pass through.

  (15) The optical separation layer has biocompatibility.

  (16) The optical separation layer is characterized in that it does not transmit biological components that degrade the detection layer.

  (17) The optical separation layer inactivates the biological component.

  (18) The method for producing the in-vivo substance measurement composition according to any one of (1) to (17), wherein a precursor for forming the optical separation layer is added to the detection layer A method for producing a composition for measuring a substance in a body, wherein the composition is combined with the detection layer.

  (19) The method for producing a composition for measuring an in-vivo substance according to any one of (1) to (17), wherein a peripheral portion of the optical separation layer is adhered around the detection layer. A method for producing a constituent for measuring a substance in a body.

  (20) The in-vivo substance measurement composition according to any one of (1) to (17), a light source that irradiates light to the detection layer from the detection layer side of the in-vivo substance measurement composition, And a photodetector that receives fluorescence from the detection layer.

  (21) The structure for measuring a substance in a body is used by contacting the living body with the optical separation layer side.

  (22) At least the light source and the photodetector are provided in the same package, and the optical separation layer side of the in-vivo substance measurement component is provided to be exposed from the outer surface of the package. .

  (23) The light source emits at least one type of wavelength, and the photodetector detects at least one type of light from the detection layer.

  According to the in-vivo substance measurement composition of the present invention, an optical separation layer that is optically opaque and transmits the substance to be measured is provided on one surface of the detection layer that emits fluorescence according to the concentration of the substance to be measured. Since the detection layer is prevented from coming into direct contact with the biological material, deterioration of the detection layer, in particular, deterioration of the fluorescent indicator in the detection layer can be suppressed, and stable measurement results can be obtained even for long-time measurement. It becomes like this. In addition, light leakage from the sensor can be prevented, and adverse effects on living tissue around the sensor can be prevented.

  In addition, according to the method for producing a composition for measuring an in-vivo substance of the present invention, the composition for measuring an in-vivo substance comprising the two-layer structure of the detection layer and the optical separation layer according to the present invention can be easily manufactured.

  Furthermore, according to the implantable substance sensor of the present invention, the use of the in-vivo substance measurement composition comprising the double structure of the detection layer and the optical separation layer according to the present invention suppresses deterioration of the detection layer, Long-term stable measurement can be performed.

  The best mode for carrying out the present invention will be described below with reference to the drawings.

  FIG. 1 is an explanatory diagram for explaining the principle configuration of a composition for measuring an in-vivo substance and an implantable substance sensor using the same according to the present invention, and FIG. 2 is a configuration in a detection layer of the composition for measuring an in-vivo substance. FIG. 3 is a conceptual diagram showing a configuration in the optical separation layer in the in-vivo substance measurement composition.

  The in-vivo substance measuring composition is used as an indicator layer in an implantable substance sensor (hereinafter simply referred to as a sensor) (hereinafter, the in-vivo substance measuring composition is referred to as an indicator layer).

  One embodiment of the indicator layer 1 to which the present invention is applied includes a detection layer 2 containing a fluorescent indicator and an optical separation layer 3 laminated on one surface of the detection layer 2 as shown in FIG. . The detection layer 2 and the optical separation layer 3 are chemically bonded to each other.

  As shown in FIG. 2, the detection layer 2 has a structure in which a fluorescent indicator 201 is supported on a detection layer base material 202. On the other hand, the optical separation layer 3 has a structure in which an opaque substance 301 is carried on an optical separation layer substrate 302 as shown in FIG.

  The indicator layer 1 is disposed so that the optical separation layer 3 faces the outer surface side of the sensor, and comes into contact with a living tissue when provided on the sensor. On the other hand, the detection layer 2 is inside the sensor and does not come into contact with the living tissue.

  In principle, the light source 4 and the light detector 5 are arranged in the structure of the sensor. By irradiating the light from the light source 4 to the detection layer 2, the fluorescent indicator 201 in the detection layer 2 becomes the substance to be measured. Fluorescence is shown according to the amount, and the light detector 5 receives the fluorescence. The photodetector 5 is a photoelectric conversion element and outputs an electrical signal corresponding to the amount of received light. The amount of the substance to be measured is measured from the output electric signal. The arrangement of the light source 4 and the light detector 5 actually varies depending on the sensor configuration.

  As the fluorescent indicator 201, any fluorescent indicator can be used as long as it is selected depending on the substance to be measured, and its fluorescent property reversibly changes in accordance with the amount of the substance to be measured. For example, in the case of a sensor that measures the in vivo hydrogen ion concentration or carbon dioxide, a hydroxypyrene trisulfonic acid derivative, a phenylboronic acid derivative having a fluorescent residue for saccharide measurement, and a fluorescent residue for potassium ion measurement. A substance containing a crown ether derivative having a group as a fluorescent substance is used.

  For the measurement of saccharides, a fluorescent substance containing a fluorescent phenylboronic acid derivative is used. Among them, as the fluorescent substance, two phenylboronic acids represented by the following chemical formula (1) and anthracene as a fluorescent residue are used. The contained compound is excellent in detection sensitivity.

(In the chemical formula (1), Q is a fluorescent residue, and at least one of R1, R2, and R3 is an active group for binding to the substrate of the detection layer; R6 and R7 are at least one substituent selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an allyl group, an allylalkenyl group, a substituted alkyl group, an oxyalkyl group, an acyl group, and a halogen atom)
Moreover, the fluorescent residue of this chemical formula (1) may be substituted. For example, naphthalene, anthracene, phenanthrene, benzanthracene, bilene, coumarin and the like can be used, and among these, anthracene is preferable because of its fluorescent properties.

  The detection layer 2 includes such a fluorescent indicator 201 and a detection layer substrate 202 that carries the fluorescent indicator 201. This is to prevent the fluorescent indicator 201 from flowing out in the sensor. It is preferable that the fluorescent indicator 201 and the detection layer substrate 202 form a bond by covalent bond formation, electrical, hydrophobic, or other interaction.

  Such a detection layer 2 can be obtained, for example, by binding a fluorescent indicator 201 having an active group to a detection layer substrate 202 having an active group that reacts therewith.

  At that time, a spacer having an appropriate length may be introduced between the two. As the spacer, an alkyl chain, a polyether chain, or the like can be used. By introducing the spacer, the mobility of the fluorescent indicator 201 is improved, thereby improving the sensor sensitivity and obtaining the effect of improving the labeling efficiency during the introduction reaction.

  As the detection layer base material 202 carrying the fluorescent indicator 201, a polymer substance having optical transparency can be used. In particular, cellulose, polyacrylamide, polyethylene glycol, and polyvinyl alcohol are preferable, but not limited thereto.

  In order to bind the fluorescent indicator 201, it is necessary that an appropriate active group exists in the detection layer base material 202. If there is no active group, it can be introduced later.

  The active group includes, for example, an amino group, a carboxylic acid group, a hydroxyl group, a halogenated carboxylic acid group, a sulfonic acid group, a halogenated sulfonic acid group, together with the active group of the fluorescent indicator 201 and the active group of the optical separation layer substrate 302. Examples thereof include a thiol group, an isocyanate group, and an isothiocyanate group.

  The fluorescent indicator 201 having an active group and the detection layer substrate 202 having an active group (and a spacer when a spacer is inserted) are optionally present in the presence or absence of a suitable solvent, catalyst, or condensing agent. Can be reacted and combined.

  When the fluorescent phenylboronic acid derivative is bonded to the detection layer substrate 202, the detection layer substrate 202 is bonded from one or both of the two nitrogen atoms shown in chemical formula (1) (R1, R2). Or by binding to a fluorescent residue (R3) or the like, and the appropriate spacer may be interposed between the fluorescent indicator 201 and the detection layer substrate 202.

  The optical separation layer 3 includes an opaque substance 301 and an optical separation layer substrate 302 that carries the opaque substance 301. The optical separation layer 3 shields excitation light from the light source 4, shields fluorescence emitted from the fluorescent indicator 201 of the detection layer 2, shields light originating from outside other than the fluorescent indicator 201, and colors in vivo It has a function of eliminating the influence of substances and fluorescent substances.

  These shielding functions not only improve the reliability of measured values, but also have an effect of preventing adverse effects on the living tissue around the sensor due to excitation light and fluorescence required for the sensor.

  The opaque substance 301 used for the optical separation layer 3 may be any substance that does not transmit the excitation light from the light source 4 and the fluorescence emitted by the fluorescent indicator 201, does not transmit light such as ultraviolet light, visible light, and infrared light, and Use non-reflective material. In particular, when ultraviolet rays are used as excitation light, it is important to prevent the ultraviolet rays from being emitted outside the sensor.

  As such an opaque substance 301, for example, carbon black, fullerene, carbon nanotube, or the like can be used. The opaque substance 301 can be supported on the optical separation layer substrate 302 by covalent bond formation or electrical or hydrophobic interaction. Further, it can be confined and supported in the polymer structure of the optical separation layer substrate 302.

  As the optical separation layer substrate 302, a polymer substance can be used, but the polymer substance may be cross-linked or modified.

  As the polymer substance, for example, dextran, polyacrylamide, polyethylene glycol, polyvinyl alcohol, polyamide and the like can be used.

  Further, as the optical separation layer substrate 302, a polymer porous film formed by a phase inversion method can be used. The phase inversion method for obtaining a porous polymer membrane for the optical separation layer substrate 302 of the present invention is a conventionally known method, and for example, a wet film forming method can be used. The wet film-forming method is a well-established technique by Loeb and Sourirajan (see Adv, Chem, Ser. 38, 117, 1963), for example, “Synthetic Polymer Membranes, a Structural Perspective Rive” R Kesting, J. Wiley and Sons).

  In this method, a polymer compound is dissolved in a solvent to form a membrane forming stock solution, and then a membrane forming stock solution formed in a desired shape is immersed in a non-solvent for the polymer compound to obtain a microporous membrane. . Cellulose derivatives, polysulfone resins, polyamide resins, aromatic polyether ketone resins and the like can be used for the polymer porous membrane. Cellulose derivatives include cellulose acetate and cellulose nitrate. Examples of the polysulfone resin include polysulfone, polyethersulfone, and sulfated polysulfone. Examples of the aromatic polyether ketone resin include polyether ether ketone and polyallyl ether ketone. The thickness of the porous polymer membrane of the present invention is preferably 5 to 100 μm, more preferably 5 to 50 μm, and further preferably 10 to 20 μm. When the thickness is less than 5 μm, which is the lower limit value, there is a problem of light shielding properties and insufficient strength.

  Moreover, the average pore diameter of the polymer porous membrane in the present invention is preferably 0.001 to 0.1 μm. More preferably, it is 0.005-0.01 micrometer. This is a value obtained by imaging and measuring the surface of the porous polymer membrane with an electron microscope, and is a smaller value on the outer side and the inner side. Moreover, when it is desired to accelerate the response speed, the average pore diameter is preferably set to 0.01 to 0.05 μm, for example.

Moreover, it is preferable that the value of glucose / bovine serum albumin of the substance permeation rate (number of permeation molecules / membrane unit area / unit time) of the polymer porous membrane in the present invention is 10 to infinity. More preferably, it is 20 to infinity, and more preferably 40 to infinity. The initial substance permeation rate was calculated from the experimental results using a diffusion cell usually used for evaluation of dialysis membranes. The experimental conditions were a single chamber volume of 50 mL, an effective surface area of the test membrane of 4.9 cm ² , and a temperature of 37 ° C. The donor cell contains a phosphate buffered saline solution of glucose 100 mg / mL (w / v) and bovine serum albumin 5% (w / v) as a simulated body fluid composition, and the recipient cell contains phosphate buffer. Only the physiological saline solution was added, and the concentration change of each component in each cell was measured at regular intervals.

  When dextran, for example, is used for the optical separation layer substrate 302, the opaque substance 301 can be retained in the dextran network structure by suspending the opaque substance 301 in a dextran solution and crosslinking.

  The optical separation layer 3 has a function of allowing the substance to be measured to pass therethrough and not allowing biological components other than at least one kind of the substance to be measured to pass. This can be obtained by controlling the molecular weight, the degree of crosslinking, and the chemical structure of the optical separation layer substrate 302 to create a gap of an appropriate size in the optical separation layer 3. In addition, a method such as drilling by a physical or chemical method may be used.

  Some biological components interfere with the interaction between the fluorescent indicator 201 and the substance to be measured and the fluorescence emission characteristics. Therefore, in the indicator layer 1, it is very useful to provide the optical separation layer 3 with a function of preventing those biological components from reaching the detection layer 2.

  For example, the optical separation layer 3 that can exclude blood cells, biological proteins and polysaccharides is preferable. In the case of a sensor that uses a sugar or ion as a substance to be measured, the optical separation layer 3 that transmits only the substance to be measured and does not transmit blood cells or proteins can be created by utilizing the difference in molecular weight.

  As the optical separation layer substrate 302, when the sensor is embedded in the body, the optical separation layer 3 itself that is the outermost surface of the indicator layer 1 does not change its physical properties on the surface due to various biological reactions. In addition, in order to reduce the influence of the indicator layer 1 on the living body, it is preferable to use a polymer substance excellent in biocompatibility.

  Examples of such a polymer substance include dextran, cellulose derivatives, polyacrylamide, polyethylene glycol, polyvinyl alcohol, cellulose, polysulfone resin, polyamide resin, and aromatic polyether ketone resin. Further, in order to improve biocompatibility, the surface of these polymer substances may be further modified or a compound may be bound.

  Furthermore, the optical separation layer 3 has a function of deteriorating the detection layer 2 and not transmitting a biological substance that may disturb the fluorescence characteristics. Specifically, for example, a radical, an oxidizing substance, a reducing substance, or a substance that promotes photolysis of the fluorescent indicator 201 that exists in the living body and may deteriorate the fluorescent indicator 201 is inactivated or adsorbed. Thus, these substances are prevented from reaching the detection layer 2. This is because, for example, when a biological substance that affects the interaction between the fluorescent indicator 201 and the substance to be measured reaches the detection layer 2, the fluorescence characteristics of the fluorescent indicator 201 may change, or the fluorescent or colored biological substance itself may change. The measurement result may be upset by reaching the detection layer 2. Therefore, such a substance is prevented from reaching the detection layer 2.

  For this purpose, a substance having these functions is applied to the optical separation layer substrate 302, or an opaque substance 301 having these functions is itself used.

  As a more specific example, it becomes possible by modifying or carrying vitamin E, polyphenols, metal chelates, etc. on the optical separation layer substrate 302. Such a function can also be obtained by using carbon black, fullerene, or a derivative thereof as one of the opaque substances 301. Further, the optical separation layer substrate 302 may be modified with an antioxidant or a radical activator, or activated carbon having an action of adsorbing an interfering substance may be used as one of the opaque substances 301.

  Thus, it is useful to obtain an accurate and reproducible measurement result by providing the optical separation layer 3 with a function of inactivating or adsorbing a biological component that affects the detection of a substance to be measured.

  Further, by providing the optical separation layer 3 with such a function, the detection layer 2 can be protected, and the detection and measurement functions of the substance to be measured can be maintained for a long time.

  Note that, as the optical separation layer 3, ideally, only the substance to be measured is selectively transmitted to the detection layer and other biological substances are not transmitted. However, in practice, it is very difficult to selectively permeate only the substance to be measured, or it is impossible depending on the substance to be measured. Therefore, in the optical separation layer 3 according to the present embodiment, as described above, measurement is performed by preventing at least one kind of biological material that inhibits the fluorescence of the detection layer or deteriorates the detection layer 2 from passing therethrough. The accuracy of the measurement is improved, and the stability of measurement over a long period can be obtained.

  The optical separation layer 3 and the detection layer 2 are preferably chemically bonded to each other. Chemical bonds refer to covalent bonds, ionic bonds, hydrophobic bonds, and the like.

  The optical separation layer 3 and the detection layer 2 are bonded via a cross-linking agent having active groups at both ends that bind the active groups present in the optical separation layer 3 and the active groups present in the detection layer 2. It can be done by methods.

  Further, a chemical bond with the detection layer 2 can be formed simultaneously with the crosslinking of the optical separation layer substrate 302 of the optical separation layer 3.

  For example, after forming the detection layer 2 first, the optical separation layer substrate 302 containing the opaque substance 301 and the crosslinking agent or a precursor thereof may be laminated on the detection layer 2 and reacted. .

  For example, when dextran is used for the substrate 302 for the optical separation layer, the dextran solution in which the opaque substance 301 is suspended is added to the detection layer 2 and then crosslinked to retain the opaque substance 301 and bind to the detection layer 2. Can be performed simultaneously.

  When a polymer porous membrane is used for the optical separation layer substrate 302, the peripheral portion of the optical separation layer substrate 302 can be adhered to the periphery of the detection layer with a resin adhesive.

  In addition, the detection layer 2 and the optical separation layer are obtained by polymerizing the polymerizable compound in the porous structure of the polymer porous membrane using a polymerization compound having an active group bonded to the active group present in the detection layer 2. A bond with 3 can also be formed. Examples of the polymerizable compound having an active group include ethylene glycol diglycidyl ether, glycidyl methacrylate, N, N′-methylenebisacrylamide, N, N ′-(1,2-dihydroxyethylene) -bisacrylamide, and ethylene glycol diester. There are methacrylate, ethylene dimethacrylate and the like.

  In addition, as an adhesive for resin used for bonding the peripheral portion, for example, an epoxy adhesive, a silicon adhesive, an acrylic adhesive, or the like can be used.

  Next, an embedded material sensor using such an indicator layer will be described.

  In principle, the sensor only needs to have the light source 4 that emits the excitation light and the photodetector 5 that receives the fluorescence as described above. An amplifier that amplifies, a microprocessor that performs signal processing after amplification, a transmitter that transmits data to the outside, and a battery are required. And since the sensor itself embeds all of the sensors in the body, all these parts must be packaged together.

  In the case of a fully embedded sensor, a light source 4 that emits light having at least one wavelength as a fluorescence detection system and a light having at least one wavelength can be detected in a package that can maintain liquid tightness. A possible photodetector 5 (photoelectric conversion element) is incorporated.

  And the indicator layer 1 which is the constituent for a body substance measurement by this invention is provided in a part of package. At this time, the optical separation layer 3 side is arranged outward so as to come into contact with the living body, the excitation light from the light source 4 is irradiated to the internal detection layer 2 side, and the fluorescence from the detection layer 2 is received by the photodetector 5. As shown, the light source 4 and the light detector 5 are arranged.

  As the light source 4, for example, a light emitting diode or a semiconductor laser can be used. And the excitation light from this light source 4 is made to irradiate the detection layer 2 with an optical fiber, a lens, a mirror, a prism, an optical filter etc. as needed. On the other hand, the photodetector 5 is a photoelectric conversion element, and uses, for example, a photodiode or a phototransistor.

  Then, the amplifier, the microprocessor, and the transmitter amplify the analog electrical signal from the photodetector 5, convert it to a digital signal, and transmit it to the outside. In order to transmit a signal to the outside, for example, an antenna is provided in a portion kept liquid-tight inside or outside the sensor and transmitted. Alternatively, the signal may be transmitted through the living body percutaneously.

  A more specific example of a fully embedded sensor will be described.

  4 is a perspective view showing the external appearance of the fully embedded sensor, FIG. 5 is a partially broken perspective view showing the internal structure of the detection device, and FIG. 6 is a sectional view taken along the line AA in FIG. 7 is an enlarged cross-sectional view of portions such as an indicator layer, an optical waveguide, and a photodetector in the cross section shown in FIG.

  This sensor has a housing 11 that is packaged so as to keep its inside liquid-tight in appearance, a window portion 12 that exposes only the indicator layer 1, and an antenna portion 13 that communicates with a system outside the body.

  The inside of the sensor is in contact with the bodily fluid in the body and senses the concentration of the substance to be measured, and is provided so as to close the window 12 in order to keep the inside liquid-tight, and the indicator layer 1 is in close contact with the sensor layer. From the transparent layer 22, the light source 23 that emits excitation light, the optical waveguide 24 that guides the light from the light source 23 to the indicator layer 1, the photodetector 25 that detects the fluorescence from the indicator layer 1, and the photodetector 25 An integrated circuit 26 that processes the signal data and a battery 27 that is an internal power source are mounted.

  The indicator layer 1 is disposed in close contact with the outer side of the transparent transparent layer 22 that does not allow liquid to pass through. The transparent layer 22 keeps the inside of the sensor liquid-tight with the housing 11 from the outside. The housing 11 is preferably made of a material having excellent biocompatibility such as titanium.

  This sensor guides the light from the light source 23 through the optical waveguide 24 serving as a light emitting unit, and irradiates the indicator layer 1 as excitation light, so that the indicator layer 1 is fluorescent according to the concentration of the substance to be measured in the body. The fluorescence is converted into an electric signal by the photodetector 25.

  The optical waveguide 24 is installed so as to be sandwiched between the indicator layer 1 and the photodetector 25. The optical waveguide 24 is, for example, an optical fiber.

  The optical waveguide 24 is provided with a concave light shielding layer 41 on the photodetector 25 side. The light shielding layer 41 is, for example, a metal pipe such as a stainless steel pipe processed so as to cover the optical detector 24 side of the optical waveguide 24, or the optical waveguide 24 side of the optical detector 25. It is the one that only plated. The inner surface 43 of the light shielding layer 41 (that is, the optical waveguide 24 side) preferably reflects light.

  The light guided from the light source 23 by such an optical waveguide 24 is not irradiated to the photodetector 25 side by the light shielding layer 41, but is irradiated only to the indicator layer 1.

  The photodetector 25 is a light receiving element that receives fluorescence from the indicator layer 1 and converts it into an electrical signal corresponding to the amount of received light.

  An appropriate distance 32 is provided between the transparent layer 22 and the photodetector 25. The space 32 is filled with air having a refractive index of approximately 1 or a gas such as nitrogen gas.

  The detection layer 2 in the indicator layer 1 receives the excitation light, exhibits fluorescence according to the concentration of the substance to be measured, and radiates it as diffused light to the surroundings. The emitted fluorescence passes through the transparent layer 22, passes through the gap, is irradiated onto the photodetector 25, and is converted into an electrical signal. At this time, the light emitted from the optical waveguide 24 is emitted only to the indicator layer 1 in the direction opposite to the photodetector 25, and as a result, the photodetector 25 is not directly irradiated with the excitation light.

  On the other hand, the surface of the transparent layer 22 on the side of the photodetector 25 is in contact with a gas having a refractive index of approximately 1, such as air or nitrogen gas. Therefore, the total reflection angle of the excitation light at the interface 44 between the transparent layer 22 and the indicator layer 1 is necessarily larger than the total reflection angle at the interface 45 of the opposite transparent layer 22, and as a result, at the interface 44. It is substantially avoided that the totally reflected excitation light is irradiated on the photodetector 25, and the photodetector 25 can efficiently receive the fluorescence component from the indicator layer 1 among the components of the irradiated light. It can be done.

  The integrated circuit 26 includes an amplifier, a microprocessor, and a transmitter. The integrated circuit 26 processes the electrical signal from the photodetector 25, temporarily stores it, and transmits it to the system outside the body from the antenna unit 13 installed on the outer surface of the housing as appropriate.

  The antenna unit 13 is provided inside the antenna unit 13 so as to be embedded in a resin or the like so that the antenna coil 28 does not come into contact with body fluid.

  In the housing 11, the transparent layer 22 is fixed around the window portion 12 of the housing 11 with an adhesive 50, and the integrated circuit 26 and the detector 25 are disposed on the spacer substrate 51. By being supported by the spacer 52, a predetermined interval 32 is maintained between the detector 25 and the transparent layer 22. As described above, by maintaining the predetermined distance 32 between the detector 25 and the transparent layer 22 by the spacer substrate 51 and the spacer 52, a soft package made of resin other than metal is used as a member for forming the housing 11. Even if there is, it is possible to always maintain the predetermined interval 32.

  The following examples further illustrate the invention.

Example 1
Creating an indicator layer for glucose measurement 1
(1) Synthesis of fluorescent indicator “9,10-bis ((N-methyl-N- (ortho-boronobenzyl) amino) methyl) anthracene-2-carboxylic acid” Synthesis of methyl-9,10-bis (bromomethyl) anthracene-2-carboxylic acid 360 mL methyl-9,10-dimethylanthracene-2-carboxylic acid, 540 mg N-bromosuccinimide, and 5 mg benzoyl peroxide were mixed with 4 mL chloroform and 4 The mixture was added to a mixture of 10 mL of carbon chloride and heated to reflux for 2 hours. After removing the solvent, the residue was extracted with methanol to obtain 430 mg of methyl-9,10-bis (bromomethyl) anthracene-2-carboxylic acid.

B. Synthesis of methyl-9,10-bis (aminomethyl) anthracene-2-carboxylic acid 400 mg of methyl-9,10-bis (bromomethyl) anthracene-2-carboxylic acid obtained in A above was purchased in 60 mL of chloroform. Then, 8 mL of a 2M methylamine methanol solution was added, and the mixture was stirred at room temperature for 4 hours. After removing the solvent, the residue was purified by a silica gel column using methanol / chloroform as an eluent to obtain 235 mg of methyl-9,10-bis (aminomethyl) anthracene-2-carboxylic acid.

C. Synthesis of 9,10-bis ((N-methyl-N- (ortho-boronobenzyl) amino) methyl) anthracene-2-carboxylic acid Methyl-9,10-bis (aminomethyl) anthracene-2 obtained in B -Carboxylic acid 200 mg, 2- (2-bromomethylphenyl) -1,3-dioxaborinane 700 mg, N, N-diisopropylethylamine 0.35 mL was dissolved in 3 mL dimethylformamide and stirred at room temperature for 16 hours. After removing the solvent, the residue was purified by a silica gel column using methanol / chloroform as an eluent to obtain 194 mg of methyl ester.

  This was dissolved in 5 mL of methanol, 1 mL of 4N sodium hydroxide was added, and the mixture was stirred at room temperature for 10 hours. Then, it neutralized with hydrochloric acid, the inorganic salt was removed by gel filtration, and 180 g of 9,10-bis ((N-methyl-N- (ortho-boronobenzyl) amino) methyl) anthracene-2-carboxylic acid was obtained.

The resulting 9,10-bis ((N-methyl-N- (ortho-boronobenzyl) amino) methyl) anthracene-2-carboxylic acid has a melting point of 121 ° C. and ¹ H-NMR data in DMSO-d ₆ is 2.15 ppm (d, 6 H, N—CH ₃ ), 4.10 ppm (m, 4 H, N—CH ₂ -benzene), 4.45 ppm (m, 4 H, N—CH ₂ -anthracene), 7.55- It was 8.90 ppm (m, 15 H, N—CH ₂ -aromatic).

  The chemical formula showing the reaction in the preparation process of the fluorescent indicator is shown below.

(2) Preparation of detection layer for glucose measurement 1.25 g of ethylene glycol diglycidyl ether was dissolved in 4 mL of dimethyl sulfoxide, and 16 mL of distilled water containing 60 mg of sodium hydroxide was added, and 10 cm × 10 cm shallow square stainless steel Placed in a container.

  A regenerated cellulose membrane (Cuprophan) cut into 10 cm square was gently immersed in this container, reacted at room temperature for 20 minutes, then the reaction solution was removed, and gently washed 4 times with 40 mL of distilled water.

  Subsequently, the sample was immersed in 20 mL of 4.2% (w / v) 1,6-hexanediamine aqueous solution in the same container, reacted at room temperature for 2 hours, then the reaction solution was removed, and 4 mL with 40 mL of distilled water. This was gently washed twice with 20 mL of dimethylformamide to obtain an activated cellulose membrane.

  9,10-bis ((N-methyl-N- (ortho-boronobenzyl) amino) methyl) anthracene-2-carboxylic acid 20 mg, 1- (3- (dimethylamino) propyl)-obtained in (1) above 12 mg of 3-ethylcarbodiimide and 8 mg of 1-hydroxybenzotriazole are dissolved in 10 mL of dimethylformamide and taken in the same container, and the activated cellulose membrane prepared by the above reaction is immersed therein.

  After reacting at room temperature for 17 hours, it was washed 3 times with 20 mL of dimethylformamide, twice with 40 mL of 0.01N hydrochloric acid and 3 times with 40 mL of distilled water, and then 50 mM phosphate buffer (pH = 7.0) For 10 hours or more.

(3) Preparation of indicator layer 7 g of dextran was stirred and dissolved in 175 mL of distilled water at 50 ° C., and 5 g of carbon black was added, followed by sonication until uniformly dispersed. Thereto, 3.5 mL of a 50% aqueous sodium hydroxide solution and 6.5 g of ethylene glycol diglycidyl ether were added, and the mixture was stirred at 45 ° C. for 30 minutes. The solution thus obtained becomes a precursor for forming the optical separation layer. At this stage, dextran crosslinking, polymerization, and functional group introduction occur in part.

  The solution was further stirred by adding 230 mL of distilled water, and the mixed solution was put into a sprayer.

  The glucose measurement detection layer prepared in the above (2) was fixed to a flat glass plate, the mixed solution was sprayed uniformly, and further dried in an oven at 45 ° C. for 30 minutes to form an optical separation layer. By this heat drying, cross-linking and polymerization inside the dextran further progress to complete a network structure and bond with the detection layer. This obtained the thin film used as the indicator layer based on this invention.

(Example 2)
Preparation of indicator layer for glucose measurement 2
A. Preparation of detection layer 8 g of perchloric acid aqueous solution (effective chlorine concentration 5%) and 5 mL of 12N sodium hydroxide aqueous solution are mixed with 15 mL of distilled water, taken into a 10 cm × 10 cm shallow square stainless steel container, and cooled to 0 ° C. did. A polyacrylamide membrane cut into a 10 cm square was gently immersed in this container and reacted at −5 ° C. for 2 hours.

  The reaction solution was removed and washed gently 4 times with 40 mL distilled water and twice with 20 mL dimethylformamide to obtain an activated polyacrylamide membrane.

  9,10-bis ((N-methyl-N- (ortho-boronobenzyl) amino) methyl) anthracene-2-carboxylic acid 20 mg, 1- (3- (dimethylamino) propyl) -3- synthesized in Example 1 12 mg of ethylcarbodiimide and 8 mg of 1-hydroxybenzotriazole are dissolved in 10 mL of dimethylformamide and taken in the same container, and the activated polyacrylamide membrane prepared by the above reaction is immersed therein.

  After reacting at room temperature for 17 hours, it was washed 3 times with 20 mL of dimethylformamide, twice with 40 mL of 0.01N hydrochloric acid and 3 times with 40 mL of distilled water, and then 50 mM phosphate buffer (pH = 7.0). For 10 hours or more.

B. Application of optical separation layer to detection layer 7 g of dextran was stirred and dissolved in 175 mL of distilled water at 50 ° C., and 5 g of carbon black was added, followed by sonication until evenly dispersed. Thereto, 3.5 mL of a 50% aqueous sodium hydroxide solution and 6.5 g of ethylene glycol diglycidyl ether were added, and the mixture was stirred at 45 ° C. for 30 minutes to obtain a precursor serving as an optical separation layer. Further, 230 mL of distilled water was added to this solution, and this mixed solution was put in a sprayer.

  The detection layer prepared in A is fixed to a flat glass plate, the mixed solution is uniformly sprayed, and further dried in an oven at 45 ° C. for 30 minutes to form an optical separation layer. The indicator layer according to the present invention A thin film was obtained.

Example 3
Preparation of indicator layer for glucose measurement 3
7 g of dextran was stirred and dissolved in 175 mL of distilled water at 50 ° C., and 5 g of fullerene was further added, followed by sonication until it was uniformly dispersed. Thereto were added 3.5 mL of a 50% aqueous sodium hydroxide solution and 6.5 g of ethylene glycol diglycidyl ether, and the mixture was stirred at 45 ° C. for 30 minutes to obtain a precursor serving as an optical separation layer. Further, 230 mL of distilled water is added to the solution, and the mixed solution is put into a sprayer.

  The glucose measurement detection layer prepared in (2) of Example 1 was fixed on a flat glass plate, the above mixed solution was sprayed uniformly, and further dried in an oven at 45 ° C. for 30 minutes to form an optical separation layer. A thin film was formed to be an indicator layer according to the present invention.

Example 4
Preparation of indicator layer for glucose measurement 4
20 g of cellulose diacetate was dissolved in 150 g of dimethylformamide, 50 g of triethylene glycol was added, and 6 g of carbon black was further added and stirred and dispersed until uniform. The obtained slurry was cast on a glass plate using an applicator adjusted to a clearance of 50 μm, solidified in a 30% DMF aqueous solution, further immersed in a 5% glycerin aqueous solution, and the film peeled off from the glass plate was collected. After sufficiently washing with a 5% glycerin aqueous solution, it was dried at room temperature for 24 hours to obtain an optical separation layer. The average pore diameter of the membrane surface observed by an electron microscope was 0.01 μm, and the glucose transmission rate / bovine serum albumin transmission rate was 71.6.

  The detection layer for glucose measurement prepared in (2) of Example 1 was fixed to a flat glass plate, and an optical separation layer cut out larger than the detection layer was laminated, and the periphery (0.5 to 1 mm from the outer peripheral edge) was laminated. Part) was fixed with an epoxy adhesive to obtain a thin film to be an indicator layer according to the present invention.

(Evaluation 1)
Evaluation of the glucose response of the indicator layer The glucose response of the indicator layer was evaluated. For comparison, an indicator layer using only the glucose measurement detection layer prepared in (2) of Example 1 (that is, the one without an optical separation layer) was used as a comparative example (hereinafter, the comparative examples are the same). is there).

  In this evaluation, the indicator layer was fixed to the evaluation apparatus, and the response of the fluorescence intensity to the glucose concentration in plasma was evaluated.

  FIG. 8 is a schematic diagram showing the structure of an evaluation apparatus for evaluating the glucose responsiveness of the indicator layer.

  This evaluation apparatus includes a flow cell 51 capable of fixing the indicator layer 1 and circulating a liquid therein, a light source 4 for irradiating excitation light onto the surface of the flow cell 51 on which the indicator layer 1 is fixed, and the indicator layer 1. And a photodetector 5 for receiving the fluorescence emission light. In an actual apparatus, the excitation light from the light source 4 is irradiated toward the indicator layer by light guided by an optical fiber from a light emitting diode or the like serving as the light source. The photodetector 5 receives the fluorescence of the indicator layer with an optical fiber and guides it to the fluorescence spectrophotometer. Here, the excitation wavelength was 380 nm and the measurement wavelength was 440 nm.

  FIG. 9 is a graph showing the results of the glucose responsiveness evaluation of the indicator layers created in Examples 1, 2, 4 and the comparative example.

  Example 1 in which the detection layer substrate of the detection layer is cellulose, Example 2 in which the detection layer substrate of the detection layer is polyacrylamide, and Example 4 in which the detection layer substrate is cellulose are the indicator layers. Both showed good glucose responsiveness, but the comparative example without the optical separation layer had almost no responsiveness.

  This is because, in the comparative example, since there is no optical separation layer, substances in plasma interfere with the interaction with the fluorescent indicator in the detection layer, and the generated fluorescence cannot be collected efficiently, or there is no optical separation layer. This is probably because light from a source other than the fluorescent indicator was picked up.

  As described above, it can be seen that in Examples 1 and 2 to which the present invention is applied, glucose can be reliably measured by using the optical separation layer.

(Evaluation 2)
Evaluation of the excitation light and fluorescence shielding effect of the indicator layer The indicator layer is fixed to the evaluation device and irradiated with excitation light of 405 nm, and the leakage of excitation light and fluorescence is measured by a photodetector arranged on the optical separation layer side of the upper surface of the sensor. did. A photodetector capable of continuously sensing light from 360 nm to 500 nm was used.

  Comparative evaluation was performed using the indicator layers prepared in Examples 1, 2, and 4 and the indicator layer of the comparative example.

  FIG. 10 is a graph showing the results of light leakage evaluation of Examples 1, 2, and 4 and the comparative example. The light leakage was shown as a relative value, with the result (measurement value) obtained by measuring the detection layer of the comparative example having no optical separation layer as 100.

  As shown in the figure, it can be seen that the indicator layers prepared in Examples 1, 2, and 4 with respect to the comparative example have almost no light leakage. This indicates that, when this indicator layer is used for an implantable sensor, the optical separation layer located outside thereof can effectively block light. Therefore, for example, when ultraviolet light or the like is used as the excitation light inside the sensor, it is possible to prevent the ultraviolet light from leaking out of the sensor and affecting the living tissue. On the contrary, it is possible to prevent the fluorescence detection from being disturbed by the light transmitted through the living tissue entering the sensor embedded in the living body.

(Evaluation 3)
Evaluation of change over time of indicator layer Only the optical separation layer side of the indicator layer is continuously contacted with plasma containing 100 mg / dl glucose at 4 ° C., and fluorescence intensity is measured over time to evaluate changes in fluorescence intensity. did.

  FIG. 11 is a graph showing the evaluation results of changes in the indicator layer over time. In addition, FIG. 11 is a graph showing how the relative fluorescence intensity has changed over time with the initial fluorescence intensity being 100%. The excitation wavelength used here is 405 nm, and the measurement wavelength is 440 nm.

  It can be seen that the indicator layers having the optical separation layers of Examples 2, 3 and 4 have less decrease in fluorescence intensity over time than the indicator layer having no optical separation layer as a comparative example. This indicates that the indicator layer having the optical separation layers of Examples 2 and 3 has a slow deterioration rate.

  Further, the indicator layer having the fullerene of Example 3 as an opaque substance in the optical separation layer has a higher fluorescence intensity over time than the indicator layer of Example 2 having the carbon black as an opaque substance in the optical separation layer. It can be seen that the decrease is small and the deterioration rate is slow.

  Such a result indicates that the optical separation layer contributes to prevention of deterioration of the detection layer, and that fullerene has a higher deterioration delaying action than carbon black. This is probably because fullerene and carbon black inactivate substances that promote deterioration in plasma, such as radicals, to suppress decomposition of the fluorescent indicator.

  From the embodiments and examples described above, the in-vivo substance measurement composition (indicator layer) according to the present invention is provided with an optical separation layer on the detection layer that emits fluorescence according to the substance to be measured. The response characteristics to the substance to be measured are improved, and the optical separation layer prevents biological substances that interfere with the measurement of the substance to be measured from reaching the detection layer, so that the substance to be measured can be reliably measured. Further, it is possible to suppress the deterioration of the detection layer, particularly the fluorescent indicator therein, and to perform continuous measurement for a long time.

  Although the embodiments and examples of the present invention have been described above, the present invention is not limited to these embodiments and examples.

  For example, in the above-described embodiments, a thin film-like indicator layer is manufactured, but the shape thereof is not limited, and may be any shape such as a rod shape or a pellet shape. Further, the size and thickness can be freely determined according to the purpose of use. In this case, the optical separation layer only needs to be provided on the detection layer so that the biological substance does not directly contact the detection layer, and the shape and size of each can be freely set independently. .

  Furthermore, it goes without saying that the present invention can be variously modified by those skilled in the art, and these modified examples are also included in the scope of the technical idea of the present invention.

  The composition for measuring an in-vivo substance of the present invention is an implantable substance sensor for measuring an object to be measured in a living body. It can be used suitably. In addition, the sensor of the present invention can be used publicly for continuously or intermittently measuring a substance to be measured in the body.

It is explanatory drawing for demonstrating the principle structure of the structure for a body substance measurement by this invention, and a sensor using the same. It is a conceptual diagram which shows the structure in the detection layer in the structure for a body substance measurement. It is a conceptual diagram which shows the structure in the optical separation layer in the structure for a body substance measurement. It is a perspective view which shows the external appearance of a fully embedded type sensor. It is a partially broken perspective view which shows the internal structure of a fully embedded type sensor. It is sectional drawing which follows the AA line in FIG. It is an expanded sectional view of parts, such as an indicator layer in the cross section shown in FIG. 6, an optical waveguide, and a photodetector. It is the schematic which shows the structure of the evaluation apparatus for evaluating the glucose responsiveness of an indicator layer. It is a graph which shows the result of glucose responsiveness evaluation of an indicator layer. It is a graph which shows the result of light leakage evaluation of an indicator layer. It is a graph which shows the evaluation result of a time-dependent change of an indicator layer.

Explanation of symbols

1 ... indicator layer,
2 ... detection layer,
3 ... optical separation layer,
4 ... light source,
5 ... photodetector,
201 ... fluorescent indicator,
202 ... base material for detection layer,
301 ... Opaque material,
302 ... Substrate for optical separation layer.

Claims (14)

A body substance measuring component provided in an implantable substance sensor that detects and measures a substance to be measured in a living body,
A detection layer containing at least one fluorescent indicator that emits fluorescence according to the concentration of the substance to be measured;
An optical separation layer that is provided on one surface of the detection layer, is optically opaque, and transmits a substance to be measured;
A composition for measuring an in-vivo substance, comprising:   The composition for measuring an in-vivo substance according to claim 1, wherein the detection layer is made of a base material to which the fluorescent indicator is bound.   The composition for measuring an in-vivo substance according to claim 1 or 2, wherein when the substance to be measured is a saccharide, the fluorescent indicator is a fluorescent substance containing phenylboronic acid. The fluorescent substance containing phenylboronic acid is represented by the chemical formula (1)

(In the chemical formula (1), Q is a fluorescent residue, and at least one of R1, R2, and R3 is an active group for binding to the substrate of the detection layer; R6 and R7 are at least one substituent selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, an allyl group, an allylalkenyl group, a substituted alkyl group, an oxyalkyl group, an acyl group, and a halogen atom) The composition for measuring an in-vivo substance according to claim 3, wherein   The constituent for measuring a substance in a body according to claim 4, wherein Q is a fluorescent residue containing anthracene.   The composition for measuring an in-vivo substance according to claim 1, wherein the optical separation layer comprises an opaque substance and a base material carrying the opaque substance.   The composition for measuring a substance in a body according to claim 1, wherein the optical separation layer is a polymer porous film formed by a phase inversion method.   The composition for measuring an in-vivo substance according to claim 6, wherein the opaque substance is made of at least one substance selected from the group consisting of carbon black, fullerene, and carbon nanotubes.   The composition for measuring a substance in a body according to any one of claims 1 to 8, wherein the optical separation layer does not transmit a biological component that degrades the detection layer.   The in-vivo substance measurement composition according to claim 9, wherein the optical separation layer inactivates the biological component. It is a manufacturing method of the constituent for body substance measurement according to any one of claims 1 to 10,
A method for producing a composition for measuring an in-vivo substance, wherein a precursor for forming the optical separation layer is added to a detection layer and then bonded to the detection layer.   It is a manufacturing method of the composition for a body substance measurement as described in any one of Claims 1-10, Comprising: The periphery part of the said optical separation layer is adhere | attached around the said detection layer, The body substance measurement characterized by the above-mentioned. Method for manufacturing a construction. The in-vivo substance measurement composition according to any one of claims 1 to 10,
A light source for irradiating light from the detection layer side of the in-vivo substance measurement composition to the detection layer;
A photodetector for receiving fluorescence from the detection layer;
An implantable material sensor comprising:   The implantable substance sensor according to claim 13, wherein the optical separation layer side of the in-vivo substance measurement component is used in contact with a living body.

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