JP2000356639A - Sensor element, and detecting method, analyzing method, analyzer and urine analysis toilet device using it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sensor element withstanding a long-term preservation and long-term use, and to provide a detecting method and device using it. SOLUTION: This sensor element for a device for quantitatively analyzing a specific component, especially an organic component or urine component uses a high molecular weight polymer including a part capable of recognizing and bonding to a specific component in its molecule in stead of an antibody molecule or enzyme conventionally used. Thereby, degradation of sensitivity during the preservation period from manufacture of the sensor element to the service beginning is suppressed to improve the measurable number of times and to elongate the measurable period. Accordingly, storage management of the sensor element is easy, replacement frequency thereof is low, and the device using it can measure the organic component or the like with high accuracy and a low cost.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an analysis method, an analyzer and a biological component analyzer using a sensor element carrying a high molecular polymer having a molecular recognition site in the molecule.

[0002]

2. Description of the Related Art With the longevity and aging of people, individual interest in health care is increasing. In particular, in recent years, self-owned health checks for the early detection of diseases and health management during or after treatment of diseases have become important themes.

[0003] Urine, blood or saliva is an important source of information on an individual's health. By quantitatively analyzing urine sugar, urine protein, urobilinogen, occult blood, blood sugar and other components, pancreatic disorders such as diabetes can be obtained. It can also test for liver, kidney, and other dysfunctions. In particular, urine has the advantage of being able to be tested non-invasively, and therefore various devices capable of supporting personal health checks by sampling and separating urine using a home, workplace or other toilet. Has been proposed (Japanese Patent Publication No. 7-84752,
JP-A-10-2850, JP-A-10-267925).

[0004] However, such devices used in homes and workplaces have different requirements from ordinary clinical test instruments and reagents. First, especially in home use, the frequency of use is expected to be about several times per day, so the life of the sensor element as a replacement part greatly affects the running cost. In other words, if the life is short, measurement 1
The cost per operation increases. In an analyzer using a sensor, an antibody having an affinity for a specific target component or the like is used, and since these are proteins, they are denatured with time and cause a decrease in sensitivity of the sensor itself.

[0005]

In particular, in the case of an immunosensor device using an antibody, in order to regenerate the sensor element and use it repeatedly, it is necessary to dissociate the antigen and the antibody after measurement. Regeneration is performed under severe conditions for protein molecules such as high salt concentrations. Under these conditions, stress is exerted on the antibody molecule, which causes collapse of the three-dimensional structure and partial decomposition, and as a result, the ability to bind to the antigen is lost and the sensitivity of the sensor element is reduced. To go.

[0006] In addition, the decrease in sensor sensitivity observed between the manufacture of the sensor element and the start of use, and the deterioration over time other than the stress caused by the above-mentioned regeneration when the apparatus is mounted are all part of the antibody molecule. It is thought to be due to the natural decomposition and collapse of the three-dimensional structure.

[0007]

SUMMARY OF THE INVENTION Such antibody degradation is unavoidable, though varying in degree. Such a problem can be solved by replacing the antibody with a molecule having specificity of molecular recognition possessed by the antibody and having a more robust basic structure. As a molecule having such characteristics, there is a high molecular polymer synthesized by a kind of template polymerization method, that is, a so-called molecular imprinting polymer. This polymer was pulverized into fine particles after polymerization, and used for solid phase extraction and the like. However, it has been found that it can be used for sensors by forming it into a thin film. In addition, because it is an artificial high polymer, it has high mechanical strength, high temperature, high pressure resistance, acid alkali resistance,
Microbial degradation is low, and it is stable for a long time.
It also has the intrinsic properties of a polymer, and can prevent a decrease in sensitivity of the sensor element.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have now found that a sensor element carrying a high molecular polymer having a molecular recognition site can withstand long-term storage and long-term use, and is particularly effective for home urinalysis devices. Was. An object of the present invention is to provide an application to a biological component, particularly a urine component, using a sensor element that can be stored for a long time and used for a long time, and to provide an apparatus for realizing the application.

The sensor element according to the present invention carries a high-molecular polymer having a molecular recognition site in the molecule.

According to the present invention, these high-molecular polymers have both specificity for a target substance and long-term stability against stress.

According to the first preferred embodiment of the present invention, the high molecular polymer used is such that the functional monomer is 4-vinylphenylboric acid, acrylic acid, methacrylic acid, acrylates, methacrylates ( N, N, N-trimethylaminoethyl methacrylate, 2-hydroxyethyl methacrylate), methacrylamides (N- (2-aminoethyl) methacrylamide), vinylpyrrolidones, acrylamides (2,6- (acrylamido) pyridine) , Styrenes, itaconic acid (methylene succinic acid), 2-vinylpyridine, 4-vinylpyridine, 4-vinylphenylboric acid, 4-
Vinyl phenol, 2- (trifluoromethyl) acrylic acid, bisacrylamide pyridine, one selected from the group consisting of vinyl imidazole, the crosslinking agent is ethylene glycol dimethacrylate, styrene divinyl benzene,
A polymer selected from one or more of the group consisting of 1,4-dibromobutane, butyl acrylate, divinylbenzene, and styrene, which is applied as a thin film on the sensor element surface by a method such as spin coating.

According to the second aspect of the present invention, the high molecular polymer used is such that the functional monomer is 4-vinylphenylboric acid, acrylic acid, methacrylic acid, acrylates, methacrylates (N , N, N-trimethylaminoethyl methacrylate, 2-hydroxyethyl methacrylate), methacrylamides (N- (2-aminoethyl) methacrylamide), vinylpyrrolidones, acrylamides (2,6- (acrylamido) pyridine), Styrenes,
Itaconic acid (methylene succinic acid), 2-vinylpyridine,
4-vinylpyridine, 4-vinylphenylboric acid, 4-vinylphenol, 2- (trifluoromethyl) acrylic acid, bisacrylamidepyridine, a plurality selected from the group consisting of vinylimidazole, the crosslinking agent is ethylene glycol dimethacrylate, One or more copolymers selected from the group consisting of styrene divinylbenzene, 1,4-dibromobutane, butyl acrylate, divinylbenzene, and styrene, which are applied as thin films on the sensor element surface by spin coating or other methods It is.

Further, the thin film polymer for realizing these embodiments can be carried on the sensor element via a linker molecule chemically or physically adsorbed or bonded to the sensor element surface. According to any of these use modes, a sensor device capable of quantifying a target substance in a sample is provided, and such a sensor device includes a biological component and a urine component,
It can be used for measurement of environmental samples dissolved or dissolved in water. Hereinafter, examples according to the present invention will be described, but the present invention is not limited to these examples.

(Example 1) Preparation of a reaction solution of a polymer for recognizing human bilirubin 200 mg of methacrylic acid, 60 mg of bilirubin and 500 mg of ethylene glycol dimethacrylate were dissolved in 10 ml of chloroform, and azobisisobutyronitrile 50 was dissolved.
After adding mg, nitrogen gas was purged.

Example 2 Manufacture of a Sensor Element for Measuring Human Bilirubin A cover glass (size 2 × 4 cm) was prepared as a support and ultrasonically washed in the order of diluted nitric acid, a neutral detergent, and ultrapure water. . A 2 nm thick Cr adhesive layer was applied on this support by magnetron sputtering,
A gold thin film having a thickness of nm was deposited. A uniform thin film of the polymer reaction solution prepared in Example 1 was formed on this surface by spin coating. Ultraviolet rays were irradiated for 10 hours in a nitrogen atmosphere to cause a polymerization reaction. Ethanol: water: acetic acid (8: 7:
Washing with the mixed solution of 5) and further washing with methanol were performed to remove the bilirubin remaining in the polymer. In this way, a sensor element carrying a polymer thin film having a bilirubin recognition site on a gold thin film was obtained. The sensor element thus manufactured can be used in combination with a sensor device based on the surface plasmon resonance method or a sensor element using a piezoelectric element to measure the bilirubin concentration in the test solution.

(Example 3) Manufacture of urine bilirubin analyzer FIG. 3 is an external view showing a state in which the apparatus of Example 1 is mounted on a toilet, and FIG. 4 is an inside of an apparatus (urine collection unit) provided with urine collecting means. FIG. The means for collecting urine at the time of urination consists of a support 1 that can be attached to the toilet and a support that can swing about a horizontal rotation axis that extends in the transverse direction of the toilet and is spaced upward from the upper surface of the rim side. Swing arm 2
And a urine collection container 3 carried on the free end of the swing arm
And a drive means 4 for swinging the swing arm to move the urine collection container between a rest position close to the inside of the toilet rim and a urine collection position located in the toilet bowl space.

The support 1 is curved along the substantially front half of the rim of the toilet bowl in plan view and is formed in a shape to cover the substantially front half, and the support is placed on the upper surface of the substantially front half of the toilet rim. This attaches the urine collecting means to the rim.

The measuring device main body (measuring unit) 5 includes means for diluting urine collected by the urine collecting means with a pH buffer, a sensor for analyzing components of urine collected by the urine collecting means,
The apparatus includes means for cleaning and regenerating the sensor, and means for controlling each means.

An operation / display panel 6 having a measurement start button and a panel for displaying a measurement result is provided at the upper part of the main body. In addition, a solution tank containing a buffer solution used for diluting urine and washing the flow path and a calibration solution containing a test substance of a known concentration, as well as a regenerating solution that dissociates the bond between the test substance and the high-molecular polymer, is also provided in the measurement unit. Prepare.

The solution tank can be separate for each solution, or can fractionate a single tank. Further, a heater and a cooler for controlling the temperature in the solution tank, the flow path, and the sensor can be provided as needed.

Further, a temperature measuring means (thermocouple or thermistor) is arranged at an appropriate position to measure the temperature at the time of measurement.
Means for correcting the signal from the sensor based on the information may be provided. Such temperature control and temperature correction
This can be performed using a microcomputer or the like provided in the control unit in the measurement unit. The urine collecting unit and the measuring unit are connected by a connecting unit 7.

In the connection part, a flow path for transporting urine collected to the measurement part, a flow path for transporting water or buffer solution for washing the urine collection part, a urine sample which has been measured by the sensor, and a surplus generated during the measurement. A channel for discharging the solution and the like into the toilet bowl, electric power for driving the urine collection unit, electric wires for passing control signals, and the like are piped and wired in a tube of an appropriate size.

FIG. 5 is a block diagram showing the internal configuration of the measuring unit. The collected urine is conveyed into the measuring section by the syringe pump 10 and diluted with the buffer in the solution tank 11 by an appropriate means. Examples of the appropriate dilution means include a flow injection analysis (FIA) that simultaneously dilutes a urine sample by flowing the urine sample into a part of a flow channel, and these are syringe pumps for measurement. Or by controlling the operation of the valve 12.

The urine thus diluted is conveyed to the sensor 14 having the sensor element manufactured in Examples 1 and 2, and the urine protein concentration is measured. After the measurement is completed, the test substance bound on the sensor element is removed, and the regenerating solution in the solution 11 is transported to the protein sensor to enable reuse, and after a certain time, the buffer solution is transported again to regenerate the regenerating solution. Discharge.

The start of the measurement is operated by the switch unit. At this time, a switch for distinguishing between “male” and “female” may be provided to change the position of the urine collection arm.
Using the signal sent from the switch as a trigger, the control unit performs a series of operations (operation of the urine collection unit, urine collection, urine transport, dilution of urine, transport of diluted urine to the sensor, regeneration of the sensor, cleaning of the sensor, urine collection unit Washing).

At the same time, a signal obtained from the sensor is compared with a signal measured using a calibration solution having a known concentration to calculate the concentration of urine components. The calculated result is displayed on the panel of the display unit and reported to the user. The calibration liquid is stored in the solution tank. The operation of the measurement (calibration) using the calibration liquid is performed in the order of transport of the calibration liquid to the sensor, regeneration of the sensor, and washing of the sensor, and the signal obtained at this time is referred to when measuring the urine component.

Here, as an example of the sensor 14, a high molecular polymer having a molecular recognition site is supported on a metal thin film capable of exciting surface plasmon resonance, and the change in the refractive index on the sensor accompanying the binding with the test substance is measured. Anything that detects can be used. Further, a type in which a similar polymer is carried on the piezoelectric element and a change in mass on the sensor accompanying the binding with the test substance is detected as a change in the resonance frequency of the piezoelectric element can also be used.

[Brief description of the drawings]

FIG. 1 is a conceptual diagram of a bilirubin recognition polymer of Examples 1 and 2 of the present invention.

FIG. 2 is a schematic view showing a state where the sensor element according to the embodiment of the present invention is mounted on a surface plasmon resonance sensor.

FIG. 3 is a view showing a state in which the urine component device according to the third embodiment of the present invention is attached to a toilet;

FIG. 4 is a diagram showing an internal structure of a urine collection unit including a urine collection unit according to a third embodiment of the present invention.

FIG. 5 is a diagram schematically illustrating a measurement unit of a urine component device according to a third embodiment of the present invention.

[Explanation of symbols]

 Reference Signs List 21 Target substance in test liquid 22 Molecular recognition site 23 Polymer polymer thin film 24 Gold thin film 25 Prism 26 Light flux 31 Support 32 Swing arm 33 Measuring device main body (measuring unit) 34 Operation / display panel 35 Connecting unit 41 Urine collection container 42 Drive unit 51 Syringe pump 52 Solution tank 53 Valve 54 Sensor

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/566 G01N 33/566 // G01N 33/545 33/545 Z F-term (Reference) 2G045 AA13 AA16 AA22 AA36 CA25 CB03 CB04 CB07 FA11 FB15 HA06 HA08 HA09 2G059 AA01 BB13 CC16 DD13 EE01 EE02 EE04 FF07

Claims (28)

[Claims] 1. A sensor element in which a base has a high molecular polymer having a molecular recognition site in its molecule which specifically binds to a target substance. 2. The sensor element according to claim 1, wherein the high molecular polymer is chemically supported. 3. The sensor element according to claim 1, wherein the high-molecular polymer is physically supported. 4. The sensor element according to claim 1, wherein the high molecular weight polymer comprises a functional monomer for molecular recognition and a crosslinking agent. 5. The functional monomer has a carboxyl group, an amino group, a hydroxyl group, and a pyridyl group required for molecular recognition expression.
The sensor element according to claim 4, wherein the sensor element has at least one species. 6. The functional monomer is 4-vinylphenylboric acid, acrylic acid, methacrylic acid, acrylates, methacrylates (N, N, N-trimethylaminoethyl methacrylate, 2-hydroxyethyl methacrylate) , Methacrylamides (N- (2-aminoethyl) methacrylamide), vinylpyrrolidones, acrylamides (2,6- (acrylamide) pyridine), styrenes, itaconic acid (methylenesuccinic acid), 2-vinylpyridine, Four-
A polymer selected from the group consisting of vinylpyridine, 4-vinylphenylboric acid, 4-vinylphenol, 2- (trifluoromethyl) acrylic acid, bisacrylamidepyridine, and vinylimidazole. The sensor element according to 4 or 5. 7. The functional monomer is 4-vinylphenylboric acid, acrylic acid, methacrylic acid, acrylates, methacrylates (N, N, N-trimethylaminoethyl methacrylate, 2-hydroxyethyl methacrylate) , Methacrylamides (N- (2-aminoethyl) methacrylamide), vinylpyrrolidones, acrylamides (2,6- (acrylamide) pyridine), styrenes, itaconic acid (methylenesuccinic acid), 2-vinylpyridine, Four-
A copolymer selected from the group consisting of vinylpyridine, 4-vinylphenylboric acid, 4-vinylphenol, 2- (trifluoromethyl) acrylic acid, bisacrylamidepyridine, and vinylimidazole. The sensor element according to 4 or 5. 8. The sensor element according to claim 4, wherein the crosslinking agent is a polymer. 9. The method according to claim 1, wherein the crosslinking agent is a polymer selected from the group consisting of ethylene glycol dimethacrylate, styrene divinylbenzene, 1,4-dibromobutane, butyl acrylate, divinylbenzene, and styrene. The sensor element according to claim 4 or 8, wherein 10. The cross-linking agent is a copolymer selected from the group consisting of ethylene glycol dimethacrylate, styrene divinyl benzene, 1,4-dibromobutane, butyl acrylate, divinyl benzene, and styrene. The sensor element according to claim 4 or 8, wherein 11. The sensor element according to claim 1, wherein the substrate supporting the high-molecular polymer has optical transparency. 12. The sensor element according to claim 1, wherein the base supporting the high-molecular polymer is a metal. 13. The sensor element according to claim 1, wherein the substrate supporting the high-molecular polymer is a metal thin film having a thickness of 100 nm or less formed on a light-transmitting support. 14. The sensor element according to claim 12, wherein the metal constituting the base is any one of gold, silver, copper, platinum and aluminum. 15. The high molecular weight polymer supported on a substrate has a thickness of 100 nm or less.
15. The sensor element according to any one of items 14 to 14. 16. A detection method for measuring, as a signal, an optical change amount resulting from a bond between a recognition site of a high molecular polymer carried on the sensor element according to claim 11 and a target substance in a sample solution. Method. 17. A detection method wherein a change in mass derived from the binding between a recognition site of a high molecular polymer carried on the sensor element according to claim 11 and a target substance in a sample liquid is measured as a signal. Method. 18. A method for analyzing a target substance in a test sample liquid, the method comprising: contacting the test substance with a test sample liquid.
An analysis method comprising a step of observing a change amount specific to each detection device by the detection method according to any one of claims 6 and 17, and a step of dissociating a bond between a polymer and a target substance on the sensor element. 19. The analysis method according to claim 18, wherein the detection method utilizes surface plasmon resonance. 20. An apparatus for analyzing a target substance in a test sample liquid, wherein the sensor element according to any one of claims 1 to 15, a detection unit using the sensor element, and a liquid sending apparatus for bringing the test sample liquid into contact with the sensor element. Analysis device comprising a part. 21. An apparatus for quantifying a target substance in a test sample solution, wherein the analysis method according to claim 18 or 19 is automatically performed, and a standard solution containing a target substance having a known concentration is analyzed. By comparing the signal in the case and the signal when the target substance in the test sample solution is analyzed,
An analyzer for calculating the target substance concentration in a test sample solution. 22. The analyzer according to claim 20, wherein the target substance is a biological component. 23. The analyzer according to claim 20, wherein the target substance is a urine component. 24. The analyzer according to claim 20, wherein the target substance is a blood component. 25. The analyzer according to claim 20, wherein the target substance is a fecal component. 26. The analyzer according to claim 20, wherein the target substance is a saliva component. 27. A support that can be attached to the toilet bowl, and a swinging mechanism that is supported by the support so as to be capable of swinging about a horizontal rotation axis that extends in the transverse direction of the toilet bowl and is spaced upward from the upper surface of the rim side portion. A moving arm, a urine collection container carried on the free end of the swing arm, and a urine collection container for moving the urine collection container between a rest position close to the inside of the toilet rim and a urine collection position located in the toilet bowl space. 22. A urine collecting means provided with a driving means for swinging a swing arm, and the analyzer according to claim 21 for analyzing a predetermined component contained in urine collected by said urine collecting means. Urine analysis toilet equipment. 28. The support body is curved along a substantially front half of the rim of the toilet bowl in a plan view and formed in a shape to cover the substantially front half, and the support body is mounted on the upper surface of the substantially front half of the toilet rim. 28. The urine separating toilet apparatus according to claim 27, wherein the urine collecting means is attached to the rim by being placed.