JP2008026302A - Fluorescence detection type chemical biosensor and method for detecting specific substance in specimen using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence detection type chemical biosensor and a detecting method that uses the same, capable of precisely detecting a specific substance in a specimen in a short time. <P>SOLUTION: The chemical biosensor is made up by immobilizing or applying a fluorescent derivatization material which does not emit fluorescence, having a detection wavelength but produces a material emitting the fluorescence, having the detection wavelength by a condensation reaction with the specific substance in the specimen, to the surface of a solid substrate. The fluorescence emitted from the material which is produced from the condensation reaction of the fluorescent derivatization material and the specific substance is detected, thereby enabling the specific substance in the specimen to be detected rapidly and with high accuracy. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a fluorescence detection type chemical biosensor and a method for detecting a specific substance in a specimen using the same, and more particularly to a fluorescence detection chemical biosensor capable of measuring at high speed and with high accuracy.

Among substances secreted in vivo, detection of chemical substances (biologically active amines) such as catecholamines and indoleamines that have aromatic or cyclic hydrocarbons and primary or secondary amines in their structures This is performed by derivatizing the product with a fluorescent derivatized substance, etc., and then separating the substance by liquid chromatography, electrophoresis, etc., and specifying the substance by fluorescence (see Non-Patent Documents 1-3, Patent Documents 1-4, etc.) ).
However, these conventional methods use liquid chromatography or electrophoresis after the reaction between the fluorescent derivatized substance and a specific substance in the sample, so that detection is not only time consuming but also can be measured at once. There is a problem that the number is limited.

On the other hand, as a biosensor for detecting a protein or the like by fluorescence, a substance that specifically binds to a specimen to be detected, for example, an antigen, an antibody, or the like immobilized directly or indirectly on a flat substrate is used. A specimen labeled with a substance is flowed on the substrate, and a specimen specifically bound to the substance is detected by fluorescence of a fluorescent substance used for labeling (see Patent Documents 5 and 6).
However, these methods require a step of labeling the specimen with a fluorescent substance, and there is a problem that the specimen cannot be detected immediately using a sensor.
“Analysis of Single Cells by Capillary Electrophoresis with On-Column Derivatization and Laser-Induced Fluorescence Detection”, S. Douglass Gilman et al., Anal. Chem. 1995, 67, 58-64. “Determinationof Femtomole Concentrations of Catecholamines by High-performance LiquidChromatography with Peroxyoxalate”, S. Higashidate et al., Analyst 1992, 117, 1863-1868. “An Invitation toBio-Analytical Chemistry”, K. Imai, Anal. Sci., 1998 , 14, 257-264. “CatecholaminesDerivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole: characterization of chemical structure and fluorescence properties”, X. Zhu et al., Anal. Chim. Acta 2003, 478, 259-269. JP-A-5-93690 Japanese Patent Laid-Open No. 5-149938 Japanese Patent Laid-Open No. 5-232020 JP 2002-286642 A JP 2001-4630 A Japanese Patent Laid-Open No. 10-311831

  The present invention has been made in view of the above circumstances, and is a fluorescence detection type chemical biotechnology that can detect a specific substance in a specimen at high speed and with high accuracy without requiring labeling treatment with a fluorescent substance. It is an object of the present invention to provide a sensor and a detection method using the sensor and analyzing it with high accuracy in a short time.

  As a result of intensive research to achieve the above-mentioned object, the inventors have made a fluorescent derivative that does not emit fluorescence at the detection wavelength but forms a substance having fluorescence at the detection wavelength by a condensation reaction with a specific substance in the specimen. By immobilizing or applying a fluorinated substance on the surface of a solid substrate to form a sensor and detecting fluorescence emitted from the substance formed by the condensation reaction of the fluorescent derivatized substance and the specific substance on the substrate, It has been found that it is possible to detect a specific substance at high speed and with high accuracy.

The present invention has been completed based on these findings, and is as follows.
1) A sensor in which a fluorescent derivatized substance that does not emit fluorescence at a detection wavelength is immobilized on or applied to the surface of a solid substrate, and the fluorescent derivatized substance is condensed on a specific substance in the specimen on the surface of the solid substrate. A fluorescence detection type chemical biosensor characterized by reacting to form a substance having fluorescence of a detection wavelength.
2) The fluorescent derivatized substance is directly adsorbed or covalently bonded to the surface of the solid substrate, or is applied on the solid substrate together with a polymer, or is adsorbed or chemically bonded to the solid substrate. 1) The fluorescence detection type chemical biosensor according to 1) above, wherein the fluorescence detection type chemical biosensor is adsorbed or chemically bonded.
3) The fluorescence detection type chemical biosensor according to 2), wherein the chemical bond includes a bond by a silane coupling agent.
4) The fluorescence detection type chemical biosensor according to any one of 1) to 3), wherein the solid substrate is formed mainly of glass, plastic, metal, metal oxide, or silicon.
5) The specific substance is a substance secreted in the body, and has an aromatic ring, a cyclic hydrocarbon or a heterocyclic ring and a primary or secondary amine in the structure thereof. One of the fluorescence detection type chemical biosensors.
6) A method for detecting a specific substance in a specimen, in which a specimen liquid is flowed on a solid substrate on which a fluorescent derivatized substance that does not emit fluorescence at a detection wavelength is fixed or coated, and the specific substance in the specimen and the fluorescence A detection method comprising: forming a substance having fluorescence with a detection wavelength by a condensation reaction with a derivatized substance, and detecting the fluorescence.
7) The fluorescent derivatized substance is directly adsorbed or covalently bonded to the surface of the solid substrate, or is coated on the solid substrate together with a polymer, or is adsorbed or chemically bonded to the solid substrate. 6. The detection method according to 6) above, wherein the detection method is adsorbed or chemically bonded.
8) The specific substance is a substance secreted in the body, and has an aromatic ring, a cyclic hydrocarbon or a heterocyclic ring and a primary or secondary amine in the structure thereof. Detection method.
9) Incident light used for fluorescence detection is irradiated from the surface on which the fluorescent derivatization material is immobilized or applied on the solid substrate, the opposite surface, or the side surface of the solid substrate. The detection method according to any one of 6) to 8) above.
10) The mass or surface concentration of the specific substance is measured from the fluorescence intensity of the substance formed by the condensation reaction between the specific substance and the fluorescent derivatized substance in the specimen. Either detection method.
11) Any one of the above 6) to 9), wherein the type of the specific substance is specified from the fluorescence spectrum of the substance formed by the condensation reaction between the specific substance and the fluorescent derivatized substance in the specimen. Detection method.

  In the conventional method, the detection pretreatment and the detection time such as liquid chromatography or electrophoresis require 20 to 60 minutes, but in the present invention, fluorescence can be detected within 5 minutes simply by flowing the sample over the sensor. .

  Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. FIG. 1 is a schematic view of a substrate structure of a fluorescence detection type chemical biosensor of the present invention, wherein 1 represents a solid substrate, 2 represents a fluorescent derivatized substance that does not emit fluorescence at a detection wavelength, or It represents a layer composed of a phosphor material and a polymer.

The solid substrate 1 in the present invention serves as a support for ensuring the mechanical strength required for a chemical biosensor.
The shape of the substrate is not particularly limited, but is preferably formed in a card shape or a disk shape.
The material for forming the solid substrate 1 is not particularly limited as long as it can function as a support for a fluorescence detection type chemical biosensor. For example, glass, quartz, ceramics, plastics, metal, metal oxidation It can be formed of a material or a material containing silicon as a main component. Such resins include polycarbonate resin, olefin resin, acrylic resin, epoxy resin, polystyrene resin, polyethylene resin, polypropylene resin, silicon resin, fluorine resin, ABS resin, urethane resin, phenol resin, polymethyl methacrylate resin, poly Examples thereof include isobutyl methacrylate resin and polyimide resin.

  The fluorescent derivatized substance used in the present invention itself does not emit fluorescence at the detection wavelength, but forms a substance having fluorescence at the detection wavelength by condensation reaction with a specific substance in the specimen. Such fluorescent derivatized substances are already well known in the detection of catecholamines and the like using the above-described conventional liquid chromatography and electrophoresis, and examples thereof include the following.

The fluorescent derivatizing substance is used by being immobilized on the surface of the solid substrate by chemical bonding such as direct adsorption or covalent bonding, or by being coated on a solid substrate together with a polymer. Alternatively, it can be indirectly immobilized on a solid substrate. That is, in the same manner as described above, a substance having high affinity with the fluorescence derivatizing substance can be adsorbed or chemically bonded to the surface of the solid substrate, and the fluorescent derivatized substance can be adsorbed or chemically bonded onto the substance.
As the method of immobilization by adsorption, the solid substrate surface having a hydrophobic surface is physically adsorbed by hydrophobic interaction with the hydrophobic fluorescent derivatized substance, or the fluorescent derivatization is performed. An ionic functional group may be introduced into the substance and electrostatically adsorbed on the charged solid substrate surface.
In addition, as a method of immobilizing by chemical bond such as covalent bond, for example, it can be chemically bonded to the solid substrate via a silane coupling agent or via another connecting molecule. Alternatively, a metal thin film such as gold or silver can be deposited on the solid substrate, and the fluorescent derivatized material into which the thiol group is introduced can be chemically bonded onto the metal thin film. Further, the surface of the solid substrate is exposed by exposing the hydroxyl group to the surface of the solid substrate by plasma treatment or ozone treatment, and condensing a polymer chain such as a polyalkyl chain or polyethylene glycol chain having an amino group or a carboxyl group at the end. It is also possible to immobilize the polymer and soak the fluorescent derivatized substance in the polymer.

Furthermore, when the fluorescence derivatizing substance is applied onto a solid substrate together with a polymer, the polymer used is one that has a high affinity with the substrate and can be applied thinly on the solid substrate. As long as it does not react with any of the specific substance capacities in the specimen, it is not particularly limited, but a highly transparent material excellent in optical properties is preferable so as not to affect fluorescence detection, for example, polymethyl methacrylate, Polystyrene, polyisobutyl acrylate and the like can be mentioned, among which polymethyl methacrylate is an example of a preferred polymer.
Also, the coating method is not particularly limited, but the spin coating method is preferable in that a polymer solution containing a fluorescent derivatizing substance can be uniformly coated in a thin film shape.

  As the specific substance that can be detected by the fluorescence detection type chemical biosensor of the present invention, any substance can be used as long as it forms a substance having fluorescence at the detection wavelength by the condensation reaction with the fluorescence derivatizing substance. Specifically, it is a substance secreted in the body, and has an aromatic ring or cyclic hydrocarbon and a primary or secondary amine in its structure. As described above, specific substances having such characteristics are already known, and specific examples include the following.

Next, a detection method using the sensor of the present invention will be described.
In the present invention, a specimen liquid is flowed on a solid substrate on which a fluorescent derivatized substance that does not emit fluorescence at the aforementioned detection wavelength is immobilized or coated, and a condensation reaction between the specific substance in the specimen and the fluorescent derivatized substance By forming a substance having fluorescence with a detection wavelength by detecting the fluorescence, the presence or absence of the specific substance in the sample can be detected.

The sample solution is prepared using a solvent that can sufficiently dissolve the specific substance to be detected, and is prepared so that the pH is about 8 using a buffer solution. Here, when using the above (Chemical Formula 1) or (Chemical Formula 2) as the fluorescent derivatization substance, in order to prevent the condensation reaction from being suppressed by replacing the fluoro group with a hydroxyl group during the condensation reaction with the specimen, Acetonitrile is mixed in an equal ratio and used for detection. Further, when the above (Chemical Formula 3) or (Chemical Formula 4) is used as the fluorescent derivatizing substance, a sodium cyanide aqueous solution is mixed and used. Furthermore, when using the above (Chemical Formula 5) or (Chemical Formula 6), an aqueous solution containing ferricyanic potassium ions is mixed and used.
After flowing the specimen liquid thus prepared on the solid substrate, the specimen liquid is allowed to stand for a while to sufficiently perform a condensation reaction between the specific substance in the specimen and the fluorescent derivatized substance. Rinse and dry.

In the method of the present invention, the fluorescence generated from the substance formed by the condensation reaction between the specific substance in the specimen and the fluorescent derivatized substance is irradiated by irradiating the sensor thus prepared with excitation light. To detect.
For example, when detecting dopamine using 4-fluoro-7-nitrobenzofurazan (NBD-F) represented by the above (Chemical Formula 1) as a fluorescent derivatizing substance, blue light of 442 nm is used as excitation light. , And the presence or absence of fluorescence having a peak at 500 to 520 nm is detected.

In the detection method of the present invention, the excitation light used for fluorescence detection is irradiated on the surface of the solid substrate on which the fluorescent derivatization substance is immobilized or applied, or on the side surface. In addition, when the solid substrate is made of a material that transmits excitation light, the opposite surface may be irradiated.
In addition, the detection of fluorescence in the present invention is preferably performed from a direction perpendicular to the incident light because it has the least influence of transmitted light, reflected light, stray light, and the like.

In the detection method of the present invention, the mass or surface concentration of the specific substance can be measured from the intensity of the fluorescence.
In this case, it is necessary to measure the fluorescence of a standard solution of a specific substance to be detected at different concentrations in advance and create a relationship (calibration curve) between the concentration and the fluorescence intensity.

  In the detection method of the present invention, the type of the specific substance present in the specimen can be identified from the fluorescence spectrum of the substance formed by the condensation reaction between the specific substance in the specimen and the fluorescent derivatized substance. .

EXAMPLES Next, although this invention is demonstrated further in detail based on an Example, this invention is not limited to these Examples.
(Example 1)
Polymethylmethacrylate (PMMA: molecular weight ˜15,000) and 4-fluoro-7-nitrobenzofurazan (NBD-F), which is a fluorescent derivatization substance, are adjusted to 5 wt% and 0.5 wt%, respectively. A sensor surface was prepared by dissolving in a mixed solvent of dichloroethane and toluene (1: 4) and applying the solution on a synthetic quartz substrate by a spin coating method.
A specimen sample was prepared by dissolving dopamine in 200 μL of 50 mM borate buffer (pH 8) at a concentration of 10 μM, and adding 200 μL of acetonitrile thereto.
A sample sample (400 μL) was dropped on the sensor surface, allowed to stand for 5 minutes, rinsed with distilled water, and dried with nitrogen gas.
On the other hand, as a comparative example, it was hung on the sensor surface and dried in the same manner as in Example 1 except that a borate buffer (pH 8) containing no dopamine was used.
Fluorescence spectrum measurement (Hitachi fluorescence spectrophotometer F-7000) was performed by irradiating the sensor surface with blue light of 442 nm. In a comparative example in which a mixed solution of borate buffer not containing dopamine and acetonitrile was dropped, Was not detected, but when a specimen sample containing dopamine was suspended, a spectrum having a peak near 520 nm could be obtained.

(Example 2)
The concentration of dopamine in the specimen sample in Example 1 was changed to 1 μM, 10 μM, 100 μM, and 1000 μM, and fluorescence spectrum measurement was performed in the same manner as in Example 1.
The result is shown in FIG. In FIG. 2, the horizontal axis represents wavelength and the vertical axis represents intensity. From the results shown in FIG. 2, it can be seen that the mass or surface concentration of dopamine can be measured from the fluorescence spectrum intensity.

(Example 3)
Methyl methacrylate-styrene copolymer (P (MMA-co-St)) and NBD-F, which is a fluorescent derivatization substance, were mixed with dichloroethane and toluene (1: 4) so that they would be 10 wt% and 0.4 wt%, respectively. The solution was dissolved in a mixed solvent and applied onto a synthetic quartz substrate by a spin coating method to produce a sensor surface.
Dopamine and histamine were dissolved in 200 μL of 50 mM borate buffer (pH 8) at a concentration of 1000 μM, respectively, and 200 μL of acetonitrile was added thereto to prepare specimen samples of dopamine and histamine.
400 μL of each sample sample was hung on the sensor surface and allowed to stand for 5 minutes. The sensor surface was rinsed with distilled water, and the sensor surface was dried with nitrogen gas.
When fluorescence spectrum measurement (Hitachi fluorescence spectrophotometer F-7000) was performed by irradiating the sensor surface with blue light of 442 nm, as shown in FIG. 3, a peak was obtained at 509 nm in the specimen sample containing dopamine, On the other hand, in the specimen sample containing histamine, a spectrum having a peak near 518 nm could be obtained.

Example 4
Methyl methacrylate-styrene copolymer (P (MMA-co-St)) and 2,3-naphthalenedialdehyde (NDA), which is a fluorescent derivatization substance, are 10 wt% and 1.0 wt%, respectively. A sensor surface was prepared by dissolving in a mixed solvent of dichloroethane and toluene (1: 4) and applying the solution on a non-fluorescent glass substrate by a spin coating method.

A specimen sample was prepared by dissolving dopamine in 400 μL of 50 mM borate buffer (pH 9.1) at a concentration of 1000 μM, and adding 100 μL of 0.01 M aqueous sodium cyanide solution thereto.
A sample sample (500 μL) was dropped on the sensor surface, allowed to stand for 10 minutes, rinsed with distilled water, and dried with nitrogen gas.
When the fluorescence spectrum measurement (Hitachi fluorescence spectrophotometer F-7000) was performed by irradiating the sensor surface with blue light of 442 nm, a spectrum having a peak near 470 nm could be obtained as shown in FIG. .

(Example 5)
The sensor surface was created by the following procedure. 5 to 7 schematically show the procedure.
(1) By binding 3-aminopropyltriethoxysilane (APS), which is a silane coupling agent, to the surface of a synthetic quartz glass substrate that has been subjected to hydrophilic treatment, the surface is rendered reactive with amino groups. (Fig. 5)
(2) Next, the substrate whose surface is activated with amino groups is immersed in a boric acid buffer (pH 8) containing 1,4-phenylene-diisothiocyanate (PDIC), and the amino groups are connected to PDIC via thiourea bonds. Are combined. (Fig. 6)
(3) After that, it is immersed in a borate buffer (pH 8) containing p-xylylenediamine (pXDA), and PDIC and pXDA are bonded via a thiourea bond to produce a sensor surface having benzylamine at the terminal. did. (Fig. 7)

The prepared sensor surface was exposed to a solution of epinephrine dissolved in 50 mM borate buffer (pH 9) at a concentration of 500 μM for 10 minutes, rinsed with distilled water, and dried. FIG. 8 schematically shows a phosphor produced on a synthetic quartz glass substrate.
In the same manner as in Example 4, the fluorescence spectrum was measured by excitation at 375 nm. FIG. 9 is a diagram showing a fluorescence spectrum, where a solid line indicates before the epinephrine reaction and a dotted line indicates after the epinephrine reaction.
As shown in FIG. 9, a fluorescence peak due to the phosphor produced by the reaction of benzylamine and epinephrine on the sensor surface was confirmed around 470 nm.

  According to the fluorescence detection type chemical biosensor of the present invention and the detection method using the same, components that are secreted in a very small amount in a living body such as catecholamines and indoleamines are detected in a short time and at a high speed. It is useful for diagnosis of hypertension and nervous system diseases.

FIG. 3 is a structural diagram of a fluorescence detection type chemical biosensor substrate. The figure which shows the fluorescence spectrum obtained as a result of carrying out the condensation reaction with the fluorescence derivatization substance NBD-F in the PMMA film | membrane with which the specific substance dopamine was apply | coated on the solid substrate. The figure which shows the fluorescence spectrum obtained as a result of carrying out the condensation reaction with the fluorescent derivatization substance NBD-F in the P (MMA-co-St) film | membrane spin-coated on the specific substance dopamine and histamine. The figure which shows the fluorescence spectrum obtained as a result of condensation reaction with the fluorescence derivatization substance NDA in the P (MMA-co-St) film | membrane by which the specific substance dopamine was spin-coated on the solid substrate. The figure which shows typically a mode that 3-aminopropyl triethoxysilane (APS) which is a silane coupling agent was combined with the synthetic quartz glass substrate surface by which the hydrophilic process was carried out. The figure which shows typically a mode that 1, 4- phenylene diisothiocyanate (PDIC) was couple | bonded through the thiourea bond with the board | substrate which the surface became active by the amino group. The figure which shows typically a mode that the sensor surface in which benzylamine exists in the terminal was produced by couple | bonding p-xylylenediamine (pXDA) through a thiourea coupling | bonding. The figure which shows typically the fluorescent substance produced | generated by reaction of benzylamine and epinephrine on a synthetic quartz glass substrate. The figure which shows the fluorescence spectrum of the fluorescent substance produced | generated by reaction of the benzylamine on the sensor surface, and epinephrine.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Solid substrate 2 Fluorescence derivatization substance or layer containing fluorescence derivatization substance

Claims (11)

  A sensor in which a fluorescent derivatized substance that does not emit fluorescence at a detection wavelength is immobilized or coated on the surface of a solid substrate, and the fluorescent derivatized substance undergoes a condensation reaction with a specific substance in a specimen on the surface of the solid substrate. Forming a substance having fluorescence of a detection wavelength.   The fluorescent derivatized substance is directly adsorbed or covalently bonded to the surface of the solid substrate, or coated on the solid substrate together with a polymer, or on a substance adsorbed or chemically bonded to the solid substrate. The fluorescence detection type chemical biosensor according to claim 1, wherein the fluorescence detection type chemical biosensor is adsorbed or chemically bonded.   The fluorescence detection type chemical biosensor according to claim 2, wherein the chemical bond includes a bond by a silane coupling agent.   The fluorescence detection type chemical biosensor according to any one of claims 1 to 3, wherein the solid substrate is formed of glass, plastic, metal, metal oxide, or silicon as a main component.   The specific substance is a substance secreted in the body, and has an aromatic ring, a cyclic hydrocarbon or a heterocyclic ring, and a primary or secondary amine in the structure thereof. The fluorescence detection type chemical biosensor described.   A method for detecting a specific substance in a specimen, wherein a specimen liquid is flowed on a solid substrate on which a fluorescent derivatized substance that does not emit fluorescence at a detection wavelength is fixed or coated, and the specific substance in the specimen and the fluorescent derivatization are detected. A detection method comprising: forming a substance having fluorescence of a detection wavelength by a condensation reaction with a substance, and detecting the fluorescence.   The fluorescent derivatized substance is directly adsorbed or covalently bonded to the surface of the solid substrate, or coated on the solid substrate together with a polymer, or on a substance adsorbed or chemically bonded to the solid substrate. The detection method according to claim 6, wherein the detection method is adsorbed or chemically bonded.   The detection according to claim 6 or 7, wherein the specific substance is a substance secreted in the body, and has an aromatic ring, a cyclic hydrocarbon or a heterocyclic ring, and a primary or secondary amine in its structure. Method.   Incident light used for fluorescence detection is irradiated from the surface on which the fluorescent derivatization material is fixed or applied on the solid substrate, the opposite surface, or the side surface of the solid substrate. The detection method according to claim 6, wherein the detection method is characterized in that:   10. The mass or surface concentration of the specific substance is measured from the fluorescence intensity of the substance formed by the condensation reaction between the specific substance and the fluorescent derivatized substance in the specimen. The detection method according to. The type of the specific substance is specified from a fluorescence spectrum of a substance formed by a condensation reaction between the specific substance and the fluorescent derivatized substance in a specimen. Detection method.

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