JP2004251740A - Bilirubin sensor utilizing mutual adsorption film - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technology capable of detecting bilirubin simply. <P>SOLUTION: A bilirubin detection membrane to be used is constituted of a mutual adsorption film wherein an aromatic amine having two or more amino groups and a spiral polymer having an ester group on the side chain are laminated as a plurality of layers alternately through a covalent bond between the amino group and the ester group on a substrate. After the membrane is subjected to a diazotization reaction and then immersed in a biosample, an ultraviolet visible absorption spectrum or a frequency change by a quartz oscillator is measured to thereby detect the bilirubin. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention belongs to the technical field of analyzing a biological sample, and particularly relates to a bilirubin sensor for detecting bilirubin contained in urine and the like.
[0002]
[Prior art]
Bilirubin is a degradation product of hemoglobin and is a major component of bile pigments. Bilirubin contained in urine is regarded as a marker of stress (tension state in a living body), and it is known that the amount increases in response to stress. In addition, if there is liver dysfunction, the blood bilirubin concentration increases, and the skin and mucous membranes become yellowish, so-called jaundice, and urinary bilirubin also increases. Therefore, the degree of liver dysfunction can be known from the amount of bilirubin.
[0003]
At present, a method mainly used for measuring bilirubin is to analyze an azo dye formed by reacting bilirubin with a diazo reagent by a colorimetric method. However, this colorimetric method does not immediately give data on the concentration and amount of bilirubin, but only prepares a standard solution within the measurable concentration range of the instrument and dilutes the test sample accordingly to perform the measurement. However, it was difficult and time-consuming.
[Patent Document 1] Japanese Patent Application Laid-Open No. 2001-49006
[Problems to be solved by the invention]
An object of the present invention is to establish a new technique capable of easily detecting bilirubin.
[0005]
[Means for Solving the Problems]
The present inventors have previously used a helical polymer as a new type of functional thin film instead of sequentially stacking polymer molecules by electrostatic interaction as in the conventionally known alternate adsorption method. An alternately adsorbed film (alternately laminated film) in which a helical polymer and a low-molecular compound are bonded by a covalent bond and alternately laminated in plurality is devised (JP-A-2001-49006: Patent Document 1). As a result of repeated studies, the present invention has been found that, if a specific low-molecular compound is used in such an alternately adsorbed film, a bilirubin sensor that can achieve the above-described object can be obtained, and the present invention has been derived. is there.
[0006]
Thus, according to the present invention, an aromatic amine having a molecular structure having at least two amino groups and a helical polymer having a molecular structure having an ester group in a side chain are formed on a substrate by the amino group and the helical polymer. Provided is a bilirubin detection thin film, comprising a plurality of alternately adsorbed films alternately stacked via a covalent bond of an ester group.
[0007]
The present invention further provides a method for detecting bilirubin in a biological sample using the above thin film,
(1) subjecting the thin film to a diazotization reaction; and (2) immersing the diazotized thin film in a biological sample and measuring an ultraviolet-visible absorption spectrum or measuring a frequency change by a quartz oscillator. And providing a method.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
The material and preparation method of the alternately adsorbed film (alternately laminated film) constituting the bilirubin sensor of the present invention are described in detail in the aforementioned JP-A-2001-49006 (Patent Document 1). A description will be given along a portion related to the embodiment of the invention.
[0009]
Helical polymer The helical polymer used in the bilirubin sensor of the present invention is an ester group which exhibits a helical structure as a whole molecule and which can form a covalent bond by reacting with an aromatic amine as described below. Is a polymer compound having a molecular structure such that has in the side chain. The preferred helical polymer to be used in the present invention is polyamino acid (polypeptide) because it is easy to handle and obtain, and it is easy to design and analyze a desired thin film. Lactic acid, nucleic acids, collagen, polysaccharides and the like can also be used. An ester group existing in the side chains of these helical polymer is not particularly limited, but generally, is represented by -COOCH ₃ or -COOC ₂ H _5. A particularly preferred helical polymer used in the present invention includes poly (γ-methyl-L-glutamate) (PMLG), which is a polyamino acid having an ester group (—COOCH ₃ ) in the side chain.
[0010]
Aromatic amine The thin film for detecting bilirubin (bilirubin sensor) of the present invention comprises a helical polymer as described above and an aromatic amine having a molecular structure having at least two amino groups to form an alternately adsorbed film. It was formed. Here, the aromatic amine having a molecular structure having at least two amino groups is generally an aromatic compound having an amino group at at least both ends or peripheral ends thereof in the molecular structure, Compounds as illustrated in FIG. 1 can be mentioned. One particularly preferred embodiment of the aromatic amine used in the present invention is paraphenylenediamine (PPDA). In the chemical structural formula shown in FIG. 1, carbon atoms and hydrogen atoms may be omitted according to a conventional expression method.
[0011]
To prepare the bilirubin detection thin Preparation <br/> present invention a thin film for bilirubin detection, the substrate was immersed in a solution of the helical polymer as described above, then, aromatic amines as described above the substrate Alternatively, the substrate is immersed in a solution of an aromatic amine as described above, and then the substrate is immersed in a solution of a helical polymer as described above. Thereafter, the operation of immersing in the helical polymer solution and the operation of immersing in the aromatic amine solution may be alternately repeated a desired number of times.
[0012]
Solvents for the solution of the helical polymer and the solution of the aromatic amine for performing such immersion operation are the solubility of the helical polymer and the aromatic amine, respectively, and the ester group and the aromatic amine in the side chain of the helical polymer. In consideration of the reactivity with water, it is appropriately selected from an organic solvent, water, or a mixed solvent of organic solvent / water. For example, PMLG is preferably an ethylene dichloride (EDC) solution, and PPDA is preferably an ethanol solution.
[0013]
The concentration of the solution of the helical polymer used in preparing the thin film for detecting bilirubin of the present invention may be lower than the concentration of the polymer solution used in the immersion operation of the conventional alternate adsorption method. The concentration of the solution of the helical polymer in the present invention is about 0.1 to 1 mM, whereas the concentration is about 100 mM to several tens of mM.
[0014]
As a material constituting the substrate (carrier) of the thin film for detecting bilirubin of the present invention, a transparent material, for example, glass, quartz, a plastic film, or the like, in the case of measuring an ultraviolet-visible absorption spectrum as a bilirubin detecting means described later. In the case where a change in frequency due to a quartz oscillator (QCM: Quartz Crystal Microbalance) is measured as a bilirubin detecting means, a metal such as gold or silver is preferable.
[0015]
These substrates are surface-treated with a suitable coupling agent so that the helical polymer or aromatic amine can be adsorbed by a first dipping operation into the helical polymer solution or aromatic amine solution. For example, when glass is used as the substrate, a helical polymer such as PMLG can be adsorbed by performing a surface treatment with a coupling agent such as 3-aminopropyltriethoxysilane. When a metal such as gold is used as a substrate, an aromatic amine such as PPDA can be adsorbed by surface-treating with a sulfur-containing coupling agent such as 3-mercaptopropionic acid. .
[0016]
After the first aromatic amine (or helical polymer) is adsorbed on the substrate as described above, alternate immersion in the helical polymer solution and the aromatic amine solution is repeated, so that the ester group in the side chain of the helical polymer is removed. An ester-amide alternating reaction occurs between the amino group of the aromatic amine and the resulting helical polymer and the aromatic amine layer, which are bonded via a covalent bond, to form an alternating adsorption film. can get. In the thin film prepared as described above, the polymer is not laminated by excessive adsorption as in the conventional alternate adsorption method, but the helical polymer is converted into an aromatic amine by an ester group present on the side of the helical structure. The aromatic amine is interposed while being covalently bonded to the helical polymer, so that the layers are sequentially laminated at a monomolecular level (ie, a unit of one polymer layer corresponding to the helical pitch of the helical polymer), so that precise film thickness control is performed. be able to. This can also be confirmed by measurement using a crystal oscillator (microbalance). Thus, the film thickness can be easily controlled according to the number of layers (the number of times of immersion), and a thin film having a thickness of several nm to several tens nm can be generally obtained.
[0017]
As is clear from the above description, in the thin film for detecting bilirubin of the present invention, the aromatic amine functions as a crosslinking agent between the layers of the helical polymer. That is, one of the at least two amino groups present in the aromatic amine reacts with the side chain of the helical polymer forming the lower polymer layer (however, the aromatic amine solution is used for the first immersion operation). In such a case, the amino group reacts with the coupling agent on the substrate) and the remaining amino group reacts with the ester group on the side chain of the helical polymer to be the next (upper) polymer layer.
[0018]
The aromatic amine used in the present invention can be further subjected to a diazotization reaction to be described later, so that a diazo group can be introduced. Must remain. Therefore, in preparing the alternate adsorption film, it is necessary to design the concentration of the helical polymer solution and the concentration of the aromatic amine in consideration of this point. The amino group does not react with the ester group in the side chain of the helical polymer, but partially reacts with the helical polymer, leaving an aromatic amine having an amino group which can be used for diazotization in the next step. For example, when PMLG is used as a helical polymer and PPDA is used as an aromatic amine, if an immersion operation is performed at a concentration of about 100 mM as an ethanol solution of PPDA with respect to 0.1 to 1 mM as an EDC solution of PMLG, an alternate adsorption film preparation After that, an unreacted amino group in an amount sufficient to undergo a diazotization reaction remains in the aromatic amine.
[0019]
Diazotization reaction To detect bilirubin according to the present invention, the alternately adsorbed film prepared as described above is subjected to a diazotization reaction. That is, as is well known, the alternating adsorption film (in which the unreacted amino groups of the aromatic amine remain) is reacted with nitrous acid at a low temperature (0 to 10 ° C.) under acidic hydrochloric acid. Introduce a group. As a result, a solid (film-like) diazo reagent in which the diazonium salt is introduced into the alternate adsorption film is obtained (see FIG. 2).
This diazotization reaction is generally performed immediately before bilirubin is detected, but if necessary, the alternately adsorbed film after the diazotization reaction can be provided as a bilirubin detection thin film according to the present invention.
[0020]
Detection of bilirubin in a biological sample According to the present invention, bilirubin in a biological sample is detected by immersing the thin film after the diazotization reaction (alternative adsorption film) in a target biological sample. can do. That is, it is known that bilirubin reacts with a diazo reagent to form two molecules of an azo dye, but in the present invention, bilirubin is converted to a diazo reagent immobilized in a thin film (an unreacted amino group is a diazo reagent). (Derived from the converted aromatic amine) and binds to the reagent, so that the change (weight change) can be detected by a quartz oscillator or spectroscopy (UV-visible absorption spectrum measurement) (see FIG. 2).
[0021]
Here, the quartz oscillator is formed by depositing metal electrodes on both sides of a thin quartz plate, and is also known as QCM (Quartz Crystal Microbalance). Using this apparatus, the weight of the substance immobilized on the surface of the metal electrode can be measured with a precision of nanograms by the change in the frequency according to the following equation.
−ΔF / F ₀ = Δm / ρAd
Here, F ₀ is the fundamental frequency, ΔF is the change in frequency, Δm is the weight of the substance on the electrode, and ρ is the density of the crystal. A is the electrode area and d is the thickness of the crystal. Using this equation, it can be seen that in a 9 MHz, AT-cut crystal resonator, the frequency of about 1 Hz decreases when 1 ng of the substance adheres to the electrode.
[0022]
When bilirubin is detected by spectroscopy (measurement of ultraviolet-visible absorption spectrum), an absorption spectrum near 445 nm is usually measured. This 445 nm is a peak derived from C = 0 of bilirubin, and the absorbance thereof increases as the amount of bilirubin bound to the diazo reagent fixed by reacting with the diazo reagent immobilized in the alternate adsorption film according to the present invention increases.
[0023]
The method of the present invention for detecting bilirubin bound to a layer of alternating adsorption through ultraviolet-visible absorption spectrum measurement or frequency change measurement by a quartz crystal oscillator (QCM) detects the amount (weight) of the bilirubin, not the concentration. However, the concentration can also be known by performing measurement under the same conditions as a sample having a known concentration and comparing. In addition, by measuring a sample from the same subject under the same conditions and tracking the relative change, it is possible to examine abnormalities and disorders in the living body (for example, the degree of stress from the relative change in the amount of bilirubin in urine). Or liver damage).
The method of the present invention using a bilirubin-detecting thin film composed of an alternately adsorbed film is performed by immediately immersing the thin film in a target biological sample and performing a quartz oscillator (QCM) measurement or a spectroscopic measurement. Knowledge about the absolute amount of bilirubin is obtained, and it is simple and does not require any complicated operation.
[0024]
Furthermore, the amount of reactive groups in the film can be freely controlled by the number of laminations (the number of times of immersion), and a highly sensitive bilirubin sensor can be obtained depending on the application. ) Is suitable for detecting bilirubin in urine.
Further, the thin film used as a bilirubin sensor in the present invention has a helical structure, and is rigid chemically and structurally because a rigid polymer is three-dimensionally cross-linked and fixed by covalent bonds. Also, the development of a disposable (disposable) bilirubin sensor that can be measured at high speed by using an alternating adsorption film that can be mass-produced at low cost is expected.
[0025]
【Example】
Hereinafter, examples will be described in order to more specifically show the features of the present invention, but the present invention is not limited to these examples.
Example 1: A micro cover glass serving as the preparation <br/> substrate detection alternate adsorption film of bilirubin using spectroscopy piranha solution _{(H 2 O 2: H 2} SO 4 = 1: those prepared in ₃₎ After washing with ultrapure water and sonic washing, nitrogen drying was performed. Next, after being immersed in a toluene solution of 3-aminopropyltriethoxysilane (1 mM) for 24 hours, washing with the toluene solution was repeated.
This glass substrate was immersed in an EDC solution (1.0 mM) of PMLG having an average degree of polymerization of 1,000 for 30 minutes to allow 3-aminopropyltriethoxysilane to react with PMLG, and then the washing was repeated with the EDC solution. Next, it was immersed in an ethanol solution of PPDA (100 mM) for 60 minutes to react PMLG with PPDA. Repeat washing with ethanol solution. This PMLG solution immersion / PPDA solution immersion operation was repeated, and the layers were stacked up to the desired number of times.
[0026]
The diazotized 0.01N HCl was diluted twice and cooled in an ice bath (sample bottle A). 0.03 mol of sodium nitrite was prepared in 10 ml of ultrapure water (sample bottle B). On the other hand, 1 mM of bilirubin was dissolved in 10 ml of chloroform to prepare a bilirubin solution. After the preparation, it was cooled to 10 ° C. or less in an ice bath.
Sample bottles A and B were immersed in an ice bath and cooled to 10 ° C. or lower. The alternately adsorbed film prepared as described above was placed in a new sample bottle C, and a necessary amount of HCl in the sample bottle A was added so that the surface was immersed. Then, both samples A and C were allowed to stand in an ice bath for 5 minutes. . The sample bottle C was taken out of the ice bath, and 0.3 ml of the aqueous solution of sodium nitrite in the sample bottle B was added into the sample bottle C. Both the sample bottles B and C were returned to the ice bath, and the sample bottle C was thoroughly stirred in the ice bath for 10 minutes so that the reaction proceeded sufficiently. The diazotized alternating adsorption film (made of a glass substrate) thus obtained was pulled up and washed with ice water.
[0027]
Spectral measurement The alternately adsorbed diazotized film was immersed in a 1 mM bilirubin solution in an ice bath prepared previously for 30 minutes (while being kept in the ice bath during the immersion). The diazotized alternately adsorbed film was taken out, washed with chloroform, dried with nitrogen, and measured for ultraviolet-visible absorption spectrum. FIG. 3 shows the measurement results.
As can be understood from FIG. 3, the absorption at the peak value of 445 nm increases with an increase in the amount of lamination. This 445 nm is a peak derived from C = 0 of bilirubin. FIG. 4 shows that bilirubin is adsorbed without performing a diazotization reaction on unreacted amino groups in the alternately adsorbed film in a state where BG (background) is matched, and bilirubin is converted after the diazotization reaction. The adsorption results are shown in comparison.
As is evident from FIG. 4, the peak value (peak absorption of C = 0 derived from bilirubin) is significantly higher when diazotization is performed on unreacted amino groups in the alternately adsorbed film. It is. From this, it can be confirmed that bilirubin is not physically adsorbed in the film, but is bound in the film by diazotizing the amino group.
[0028]
Example 2: Detection of bilirubin in urine As in Example 1, an alternate adsorption film (20 layers) composed of PMLG and PPDA was prepared and subjected to a diazotization reaction, and then bilirubin in urine was detected. did. However, as described below, a gold electrode was used as a substrate, and bilirubin was detected by frequency change measurement using a quartz oscillator (QCM).
First, as a pretreatment, a few drops of aqua regia were dropped on a 9-MHz ATcut gold-evaporated crystal oscillator (purchased from USI) to etch the electrode surface, and then washed with methanol. This crystal oscillator was modified by immersing it in a 3-mercaptopropionic acid (MPA) methanol solution (concentration: 101 mM) for 10 minutes. Then, it was immersed in a pure methanol solution for 10 minutes to wash the excess. Next, after immersion in an ethanol solution of PPDA (concentration: 100 mM) for 60 minutes, the excess was washed with ethanol. After being removed from the PPDA solution, it was immersed in a PMLG EDC solution (concentration: 1 mM) for 30 minutes. This immersion in the PPDA solution / immersion in the PMLG solution was repeated 20 times to obtain a desired alternately adsorbed film, which was then subjected to a diazotization reaction in the same manner as in Example 1.
Using the actual urine of a hepatitis C patient and a sample obtained by diluting the urine twice or five times as a sample, the above-mentioned diazotized alternating adsorption film is immersed in the sample, and the frequency change of the quartz oscillator is measured by using the Quartz Crystal. Bilirubin in urine was detected by measurement under a nitrogen atmosphere using a Microbalance (QCM) measuring device (CR05-P manufactured by USI (Inc.), Universal Counter SC-7201 manufactured by Iwasaki Communication Equipment Co., Ltd.). The result is shown in FIG. As shown in FIG. 5, it is understood that the weight of bilirubin that reacts with the membrane is detected with high sensitivity, depending on the concentration of bilirubin in urine, as measured by QCM.
[Brief description of the drawings]
FIG. 1 illustrates a chemical structural formula of an aromatic amine used in an alternate adsorption film constituting a bilirubin sensor of the present invention.
FIG. 2 schematically shows the principle of a method for detecting bilirubin according to the present invention.
FIG. 3 shows a measurement result of an ultraviolet-visible absorption spectrum when a bilirubin solution is detected using a diazotized alternating adsorption film according to the present invention.
FIG. 4 shows a comparison between the results of measurement of an ultraviolet-visible absorption spectrum when detecting a bilirubin solution using a diazotized alternating adsorption film according to the present invention and a case where a diazotized reaction-free alternate adsorption film is used for comparison. Shown together.
FIG. 5 shows an example in which bilirubin in urine is detected by frequency change measurement by QCM using a diazotized alternating adsorption film according to the present invention.

Claims (5)

An aromatic amine having a molecular structure having at least two amino groups and a helical polymer having a molecular structure having an ester group in a side chain are formed on a substrate via a covalent bond between the amino group and the ester group. A bilirubin detecting thin film comprising a plurality of alternately adsorbed films alternately stacked. 2. The thin film for detecting bilirubin according to claim 1, wherein the helical polymer is a polyamino acid. The bilirubin detection thin film according to claim 2, wherein the polyamino acid is poly (γ-methyl-L-glutamate), and the aromatic amine is paraphenylenediamine. A method for detecting bilirubin in a biological sample using the thin film according to any one of claims 1 to 3,
(1) subjecting the thin film to a diazotization reaction; and (2) immersing the diazotized thin film in a biological sample and measuring an ultraviolet-visible absorption spectrum or measuring a frequency change by a quartz oscillator. Performing the method. The method for detecting bilirubin according to claim 1, wherein the biological sample is urine.

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