JPH0627025A - Molecule recognizing function film and sensor employing it - Google Patents

Abstract

PURPOSE:To obtain a molecule recognizing function film, and a sensor employing the film, for detecting variation of concentration of specific molecule by converting into variation of secondary harmonic generating power. CONSTITUTION:A molecule recognizing function film 1 is formed on a transparent substrate 2 and disposed such that a sample 5 flowing through a channel 4 in a transparent flow cell 3 touches the film 1 directly. Light (basic wave) emitted from a light emitting element 6 transmits through the flow cell 3 and the sample channel 4 and impinges on the film 1. Light transmitted through the film 1 enters into a cut filter 7 where wavelengths other than second harmonic are cut off and only second harmonic impinges on a light receiving element 8. Variation of secondary harmonic is detected and processed at a signal processing section 9 thus determining concentration or specific element contained in the sample 5.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a molecular recognition functional film for measuring the concentration of organic and inorganic compounds or bio-related compounds by generating second harmonics, and a sensor using this film.

[0002]

2. Description of the Related Art The living body has the ability to recognize inorganic substances and organic substances with high selectivity. There is known a biosensor that quickly and easily detects a specimen in a sample solution by using a functional molecule having high selectivity, or a living body itself including cells and tissues as a substance selection functional site.

The function of the sensor is divided into a part for selectively recognizing and reacting a specific sample, and a part for capturing a change in conductivity, heat generation, luminescence, etc. of the sample due to this reaction and converting it into a signal. You can think about it. As biopolymers that recognize and react with specimens, proteins such as enzymes, antibodies, and receptors are known, and these are dispersed and immobilized in a membrane composed of natural or synthetic polymers. Used. On the other hand, electrodes such as an oxygen electrode, a hydrogen peroxide electrode, an ion electrode, and a gas electrode are generally used in a portion for capturing a reaction and converting it into a signal. Also, recently, many sensors and the like utilizing the light emission of the above-mentioned specimen have been proposed.

The electrochemical measuring method using the above electrode is
There are drawbacks such as (1) electrical and magnetic noises are likely to occur during measurement, and (2) a small amount of sample cannot be measured because a reference electrode is used. On the other hand, the optical measurement method is disclosed in, for example, JP-A-61-292045, JP-A-63-75542, and JP-A-63-271162. Sensitive light detection is now possible,
(2) Electric and magnetic noises are less likely to occur during measurement, (3) No reference electrode is required, and other advantages.When an optical fiber is used for light incidence and detection, the reaction field Since it is not necessary to physically contact the sample with the optical fiber, it is easy to exchange reagents in the reaction field, and there is the possibility that multiple samples can be measured simultaneously using multiple wavelengths. is doing.

On the other hand, as a material for generating the above-mentioned second harmonic, an organic nonlinear optical material is theoretically studied because it may possibly realize higher nonlinear optical characteristics than an inorganic material. In order to realize high second harmonic generation (SHG), it is necessary to arrange molecules having a high molecular polarizability in an arrangement structure (asymmetric orientation) having no inversion center. For this reason, research that was initially conducted on single crystal materials has recently been conducted on cumulative films having an asymmetrically oriented structure formed by the Langmuir-Blodgett (LB) method (Applied Physics No. 60). Volume 6 (1991) P586). This LB method
(1) Easy formation of an array structure without a reversal center (asymmetric orientation), (2) always obtainable as a thin film having an optical axis in the normal direction of the substrate, (3) control of thin film on a molecular level basis It is possible to do, (4) by introducing a polymerizable group, it is possible to pattern by using the lithography technology, (5) it is possible to introduce a substance such as a heavy metal to control the refractive index, Moreover, when a molecule having a high molecular polarizability is used as a film forming component, L formed
The B film itself also has high polarization characteristics, and thus has a great feature that it exhibits high second harmonic generation.

The above-mentioned second harmonic generation is one of the characteristics of the non-linear optical material, but if the molecular arrangement or the molecular structure of the LB film is changed, the non-linear optical characteristics are also affected. There is an example of applying this principle to conduct a protonation reaction of LB film forming components with hydrogen chloride gas and analyzing the second harmonic change at that time (Electrochemical Society '91 Spring Annual Meeting 2A22).

[0007]

Although the above-mentioned conventional LB method can generate the second harmonic, it is still in the stage of research on how to use it as a sensor and has been put to practical use. Absent. Even in the research in which the above-mentioned change in molecular structure and non-linear optical characteristics were linked, this technique was not suitable for use as an analytical instrument because the LB film constituent molecule undergoes a direct chemical change.

[0008]

In order to solve the above problems, the present invention provides a cumulative film by introducing a compound having a molecular recognition function and a compound generating a second harmonic into the film forming components of a monomolecular film. Form. Therefore, since this cumulative film has a molecular recognition function, the three-dimensional structure of the molecular arrangement changes due to adsorption with specific molecules, reaction catalysis, etc., and the second harmonic generation function changes accordingly. Can be detected to measure the concentration of the specific molecule. The compound having the molecular recognition function and the compound that generates the second harmonic may exist in different monomolecular film forming components. In this case, two monomolecular films are alternately stacked. This makes it possible to easily form a cumulative film having an asymmetric structure.

The compound generating a second harmonic can be enhanced in its ability to generate a second harmonic by using a compound having a chromophore sandwiched between an electron donating group and an electron accepting group. If the monomolecular film contains a compound having a hydrophilic group and a hydrophobic group, a cumulative film can be easily formed by the LB method.

The molecular recognition functional film according to the present invention can be used as a sensor because it can detect the change of the second harmonic due to the influence of the specific molecule and measure the concentration of the specific molecule as described above. You can The sensor of the present invention may be constructed by diverting a commonly used maker fringe method or wedge method (“Optical Measurement Handbook” Asakura Shoten IV-1.2.1. 494 page) to generate a fundamental wave. Generated from the above molecular recognition function film, consisting of a light emitting element that is incident on the molecular recognition function film, a light receiving element that receives the second harmonic generated from this, and a signal processing unit that processes the signal from this light receiving element. It is possible to measure the concentration of the second harmonic generation characteristic of the measurement sample.

[0011]

In the molecular recognition function film of the present invention, the three-dimensional structure of the molecular arrangement changes due to adsorption with a specific compound, reaction catalysis, etc., and the second harmonic generating function also changes accordingly. Can be detected to measure the concentration of the specific compound.

[0012]

EXAMPLES Examples of the present invention will be described below. FIG. 1 is a conceptual diagram showing a sensor according to the present invention. The molecular recognition functional film 1 is formed on a transparent substrate 2 and has a transparent flow cell 3
The measurement sample 5 flowing through the internal flow path 4 is directly the molecular recognition functional film 1
It is installed to touch. The light (fundamental wave) emitted from the light emitting element 6 passes through the flow cell 3 and the flow path 4 through which the measurement sample 5 flows, and irradiates the molecule recognition functional film 1. The light including the second harmonic transmitted through the molecular recognition function film 1 is cut into wavelengths other than the second harmonic by the cut filter 7 and enters the light receiving element 8. The change of the second harmonic detected in this way is processed by the signal processing unit 9 to calculate the concentration of the specific sample contained in the measurement sample 5.

2 to 5 show a molecular recognition functional film 1 of the present invention.
And its constituent molecules. The molecular recognition functional film 1 of FIG. 2 (a) is obtained by stacking a monomolecular film A composed of molecules 10 and 11 and a monomolecular film B composed of molecules 10 and 12 in a hetero Y-type structure described later. Is. These monomolecular films can be easily made into a cumulative film by the LB method. Although only two layers are shown in the figure, the number of layers is not limited to two.

Molecules 10 and 11 have the structure shown in FIG. That is, the molecule 10 is composed of a hydrophobic group 13 and a hydrophilic group 14 as shown in FIG.
5, hydrophilic group 16 and chromophore 17. The hydrophobic group 15 in the molecule 11 also serves as an electron-donating group, and the hydrophilic group 16 also serves as an electron-accepting group. Of course, the hydrophobic group and the electron-donating group, and the hydrophilic group and the electron-accepting group are different groups. Good. Also the molecule 10
The hydrophobic group 13 and the hydrophilic group 14 used for may be any known groups, but in consideration of affinity with the molecule 11, they are the same as the hydrophobic group 15 also serving as an electron donating group and the hydrophilic group 16 also serving as an electron accepting group, respectively. Alternatively, it is preferable to use the same kind. The hydrophobic group 15 also serving as an electron donating group is preferably an alkyl group or an aryl group, and the hydrophilic group 16 also serving as an electron accepting group is preferably —COOM group, —SO ₃ M group or the like (M is a hydrogen atom or an alkali metal atom or Represents an alkaline earth metal atom).

As the polar chromophore 17, a nitro group,
Known substances such as an azo group and a conjugated diene can be used, but the chromophore 17 is preferably sandwiched between an electron donating group and an electron donating group. From the above, examples of the molecule 10 include compounds such as arachidic acid, barium stearate, stearyl alcohol, stearyl mercaptan, and stearyl amine, and examples of the molecule 11 include compounds shown in (Chemical Formula 1) to (Chemical Formula 5). Can be mentioned. The molecule 12 is a compound having a function of identifying a specific molecule and receiving its action. As the molecule 12, for example, as a compound that adsorbs a specific molecule, a crown ether having a function of adsorbing only an ion having a specific outer shape can be used, and an enzyme that catalyzes a reaction of only a specific substrate can also be used.

[0016]

[Chemical 1]

[Chemical 2]

[Chemical 3]

[Chemical 4]

[Chemical 5]

FIG. 4 is a schematic diagram showing a type of accumulation of a monomolecular film by the LB method. In this figure, (b) is a stable film having a symmetric structure (Y-type structure) in which hydrophilic groups (or hydrophobic groups not shown in the figure) are in contact with each other. However, since the structure of this cumulative film is symmetrical, the film as a whole does not polarize, and therefore the second harmonic generation capability is poor. on the other hand,
(a) (X-type structure) and (c) (Z-type structure) are asymmetric structures in which all the molecules in the accumulated monomolecular film are oriented in one direction. Therefore, the entire film is polarized due to the effect of the electron-donating group and the electron-accepting group in the molecule, and the second harmonic can be efficiently generated. However, unlike the symmetric structure of (b), the hydrophilic group and the hydrophobic group are in contact with each other, so that the film is considerably unstable. On the other hand, the structure of (d) is an accumulation of two kinds of monomolecular films alternately, and when viewed in each kind of film unit, all the molecules have an asymmetric structure in which the molecules are oriented in one direction (hetero). Y
Type structure). This hetero Y-type structure is a stable film because it has a structure in which hydrophilic groups are in contact with each other (and hydrophobic groups are also not shown in the figure), and it is the most preferable asymmetric structure according to the present invention. is there.

In the case of forming a cumulative film having the above-mentioned hetero Y-type structure, for example, as shown in FIG. 2A, the monomolecular film A is composed of a molecule 10 having a hydrophobic group and a hydrophilic group and an electron donating group. , A molecule 11 having an electron accepting group and a chromophore. Therefore, the monomolecular film A becomes a film having a second harmonic generation function. The monomolecular film B is composed of a molecule 10 having a hydrophobic group and a hydrophilic group and a molecule 12 having a function of discriminating a specific molecule and receiving its action. That is, the monomolecular film B becomes a molecular recognition functional film. On top of this monolayer B, monolayer A, monolayer B, monolayer A, monolayer B, ...
To form a cumulative film, the molecules in the plurality of monolayers A all have hydrophilic groups (electron accepting groups) facing upward, and the molecules in the plurality of monolayers B all have hydrophilic groups. Facing downward (when the molecule 12 is an enzyme, the enzyme is often hydrophilic, so it is lined up on the side of the hydrophilic group of the molecule 10. When the molecule 12 is hydrophobic, an alkyl group or the like is added. It can also be arranged on the side of the hydrophilic group). Therefore, the film as a whole has an asymmetric structure, and the monomolecular film A has a structure in which flat cells are just connected in series, and the upper surface of the film is polarized to − and the lower surface of the film is polarized to +, so that the second harmonic is generated. It can be done efficiently.

When the above membrane is used as a sensor,
Although the mechanism is not clear, a compound having a molecular recognition function in the molecule 12 expands due to the action of a specific molecule,
It is considered that deformation such as distortion occurs, and this is transmitted to the chromophore-containing molecule 11 of the monomolecular film A and affects the directionality of the molecule 11 and the like. In any case, the action of the specific molecule causes a change in the inherent second harmonic generation function, and since this change correlates with the concentration of the specific molecule, it is possible to use this to measure the concentration of the specific molecule. It will be possible.

FIG. 2 (b) is an example in which one kind of monomolecular film has both a second harmonic generation function and a molecular recognition function. When the molecule 12 having a molecular recognition function is hydrophilic, it can be immobilized in the monomolecular film by the molecule 10 having a hydrophilic group and a hydrophobic group as shown in this figure. In order to form a cumulative film by the LB method, there is a method of stacking this one kind of monomolecular film, but another kind of monomolecular film composed of molecules having a hydrophilic group and a hydrophobic group such as molecule 10 is used. Then, the hetero Y-type structure is easier to form and has better stability.

FIG. 2 (c) shows an example in which one kind of film has both a second harmonic generation function and a molecular recognition function. Since the molecules 12 having a molecular recognition function are hydrophobic in this film, when the monomolecular film is formed on the water surface, it is in the form of riding on the hydrophobic group side of the film. This film can also be easily made into a cumulative film by using the LB method. Also in this case, as in the case of (b) above, it is better to use another type of monomolecular film composed of molecules having a hydrophilic group and a hydrophobic group such as molecule 10 to form the hetero Y-type structure. It is easy to form and has good stability.

A cumulative film by the LB method in which two kinds of films are alternately stacked can be formed by using, for example, a two-tank Langmuir trough 18 as shown in FIG. The two-tank Langmuir trough 18 is provided with a tank (trough) I and a tank (trough) II for forming a monomolecular film. The tanks I and II are filled with water, and the compositions of the monomolecular films A and B shown in FIG. 2A, for example, are dissolved in an organic solvent and dropped on the respective water surfaces. This organic solvent volatilizes and only the components of each monomolecular film are developed as a gas film. Each monomolecular film thus formed is compressed by a barrier (not shown) and compressed to a desired surface pressure to form a condensed film.

The portions of the transparent substrate 2 where the film is to be formed are submerged in the tank I as shown in the figure with the monomolecular films formed in the tanks I and II as described above. At this time, the monolayer A is the transparent substrate 2
The barrier is moved so that the surface pressure does not change even when the barrier is moved to. The transparent substrate 2 used in the present invention may be any one as long as it is transparent and is not deteriorated by a sample, a solvent or the like, and examples thereof include those formed of an inorganic material such as a glass plate or an organic polymer such as an acrylic plate. Next, the flexible gates 19a and 19b with the transparent substrate 2 being sunk
To move to tank II. The transparent substrate 2 moved to the bath II is shown by an imaginary line. The tank III between the flexible gates 19a and 19b is an intermediate tank provided to prevent turbidity of the two kinds of monomolecular films. By pulling up the film forming portion of the transparent substrate 2 transferred to the bath II, the monomolecular film B is superposed on the monomolecular film A. This time, the flexible gates 19b and 19a with the transparent substrate 2 being pulled up
To return to the bath I to sink the film forming portion. A cumulative film can be obtained by repeating the above.

Specific examples of the present invention will be described below. Example 1 As the transparent substrate 2, a transparent glass plate having a refractive index nd = 1.523 was washed and used. The two-tank Langmuir trough 18 shown in FIG. 5 was used, and in tank I, the compound of (Chemical formula 1) (compound containing a chromophore, which belongs to molecule 10 of FIG. 3) and arachidic acid (molecule 10 of FIG. 3) were used. A compound having a hydrophobic group and a hydrophilic group to which CH ₃ (CH ₂ ) ₁₈ COOH) is dissolved in chloroform to form a monomolecular film A, and the surface pressure is 25 mN.
Compressed to / m. On the other hand, in tank II, glucose oxidase (a compound having a molecular recognition function, which belongs to molecule 12 in FIG. 2) and an enzyme immobilizing agent DPPE (a compound having a hydrophobic group and a hydrophilic group, which belongs to molecule 10 in FIG. 3) are contained in tank II. . Dipa
A solution of lmitoylphosphatidylethanolamine (manufactured by Sigma) in chloroform was added dropwise to form a monomolecular film B, and the surface pressure was compressed to 8 mN / m. As the enzyme and the enzyme immobilizing agent, various known compounds other than the above can be used.

Then, according to the above-described cumulative film forming method, an LB film having a hetero Y-type structure in which a total of 81 monomolecular films A and B were alternately laminated was formed on the transparent substrate 2. Then, only one surface of the transparent substrate 2 was exposed to chloroform to remove one surface of the formed film. By using the film formed on the transparent substrate 2, first, the second harmonic intensity is measured without being processed,
Next, the membrane surface is exposed to pure water for 5 minutes and then measured.
The measurement was performed after exposing the membrane surface to a glucose aqueous solution having a concentration of 0 mg / ml for 5 minutes. As a result, the second harmonic intensity was almost the same when untreated and when treated with pure water.
When it was contacted with an aqueous glucose solution, it showed a clear decrease in strength. In addition, the measurement of the second harmonic intensity is performed by irradiating the transparent substrate 2 on which the above film is formed from the film side by using the YAG laser fundamental wave (λ = 1.064 μm) to cut the fundamental wave in the transmitted light by infrared rays. It was detected by removing with a filter.

Example 2 First, two transparent glass plates having a refractive index of nd = 1.523 were prepared as the transparent substrate 2, and one of them was coated with C by a vacuum deposition method.
An r film (2 nm) was formed. The two-tank Langmuir trough 10 shown in FIG. 5 is used, and in tank I, the compound of (Chemical formula 2) (compound containing a chromophore belonging to molecule 11 of FIG. 3) and crown ether (belonging to molecule 12 of FIG. 2) are used. A compound having a molecular recognition function. Bis (12Crown4)) and arachidic acid (a compound having a hydrophobic group and a hydrophilic group belonging to molecule 10 in FIG. 3, CH ₃ (CH ₂ ) ₁₈ COOH) dissolved in chloroform are added dropwise. To form monolayer A, surface pressure 23mN / m
Compressed to. On the other hand, in tank II, the compound of (Chemical formula 2) (Chemical formula 3)
The same composition as the composition of the tank I was used except that the compound of Example 1 was used, the solution was dissolved in chloroform, and this solution was added dropwise to form a monomolecular film B. The surface pressure was also adjusted to 23 mN / m. As the above-mentioned crown ether, various other known ones can be used. However, in the case of the bicyclic one as in the present example, the structure of the crown ether is twisted when a specific ion is adsorbed, and thus the membrane is formed. Since the physical change of the structure becomes remarkable, the change of the second harmonic intensity also becomes large, which is preferable.

First, the glass transparent substrate 2 having a Cr film formed on one surface thereof was lowered into a bath II and raised in a bath I, and this operation was repeated 10 times to form 20 hetero Y-type films. Then, the surface opposite to the surface on which the Cr film had been formed was exposed to chloroform to remove the film formed on this surface. On the other hand, the glass transparent substrate 2 on which the Cr film was not formed was first lowered in the tank I and moved up in the tank II, and this operation was repeated 10 times to form 19 layers of hetero Y-type films. Then, the one-sided film formed by exposing one side to chloroform was removed. Then, the films-removed surfaces of these two substrates were bonded to each other to complete the substrate according to the present invention in which the films were provided on both surfaces.

Next, using the same YAG laser as in Example 1, the second harmonic intensity generated while rotating the substrate (that is, while changing the incident angle) was measured to obtain the fringe pattern shown in FIG. . The reason why such a pattern occurs is that the second harmonics generated in the respective films interfere with each other, and the reason that it changes depending on the incident angle is that the path length changes. Therefore, there was no change when the above-mentioned substrate was fixed at an angle at which the secondary harmonic had a minimum value and exposed to pure water for 5 minutes and then the secondary harmonic intensity was measured. Then 2 mol
When it was exposed to a sodium chloride aqueous solution having a concentration of 1 / l or 10 mol / l for 5 minutes and then measured, a clear increase in the second harmonic intensity was observed in correlation with the change in the concentration.

Comparative Example 1 A cumulative film was formed on the glass transparent substrate 2 in the same manner as in Example 1 except that glucose oxidase was not added to the composition for forming the monomolecular film B, and the second harmonic intensity was measured. As a result, no change in strength occurred even when the membrane surface was treated with a glucose aqueous solution having a concentration of 100 mg / ml.

Comparative Example 2 A cumulative film was formed on the glass transparent substrate 2 in the same manner as in Example 1 except that the compound of Chemical formula 1 was not added to the composition for forming the monomolecular film A. When the wave intensity was measured,
No secondary harmonics could be detected in any of untreated, pure water treated, and 100 mg / ml glucose aqueous solution treated.

[0031]

As described above, according to the present invention, the adsorption function of a specific compound, the complex formation function with this compound, or the catalytic action of this compound such as an enzymatic reaction or an immunoreaction,
Since various actions can be converted into changes in the intensity of the generated second harmonic to be detected, the concentration of the specific molecule can be easily detected.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a sensor according to the present invention.

FIG. 2 is a diagram schematically showing a part of a molecular recognition functional film according to the present invention.

FIG. 3 is a diagram schematically showing a part of component molecules constituting the molecular recognition functional film according to the present invention.

FIG. 4 is a diagram schematically showing a cumulative type of the molecular recognition functional film according to the present invention by the LB method.

FIG. 5 is a schematic view of a two-tank type Langmuir trough.

FIG. 6 is a correlation diagram between the incident angle of the fundamental wave and the intensity of the generated second harmonic.

[Explanation of symbols]

1 ... Molecular recognition functional film, 2 ... Transparent substrate, 3 ... Flow cell,
4 ... Flow path, 5 ... Measurement sample, 6 ... Light emitting element, 7 ... Cut filter, 8 ... Light receiving element, 9 ... Signal processing unit, 10, 1
1, 12 ... Molecule, 13 ... Hydrophobic group, 14 ... Hydrophilic group, 15 ...
Hydrophobic group also serving as an electron-donating group, 16 ... Hydrophilic group also serving as an electron-accepting group, 17 ... Chromophore, 18 ... Two-tank Langmuir trough, 19a, 19b ... Flexible gate.

Claims (4)

[Claims] 1. A catalytic function such as an enzymatic reaction with a specific compound,
In a molecular recognition functional film in which a molecular recognition substance having an adsorption function with this compound or a complex formation function with this compound is immobilized on a carrier, the film is a cumulative film in which monomolecular films are stacked in an asymmetric structure, A molecular recognition functional film, wherein at least one of a plurality of monomolecular films constituting the cumulative film contains a compound that generates a second harmonic. 2. The molecular recognition functional film according to claim 1, wherein the compound generating the second harmonic is a compound having a chromophore sandwiched between an electron donating group and an electron accepting group. 3. The molecular recognition functional film according to claim 1, wherein the molecular recognition functional film is a cumulative film in which one or more monomolecular films containing a compound having a hydrophilic group and a hydrophobic group are stacked. 4. A cumulative film in which monomolecular films are stacked in an asymmetric structure, and at least one of a plurality of monomolecular films constituting the cumulative film contains a compound that generates a second harmonic. A molecular recognition functional film, a light emitting element that generates a fundamental wave and enters the molecular recognition functional film, a light receiving element that detects a second harmonic generated from the molecular recognition functional film, and a signal processing unit that processes a signal from the light receiving element. Sensor consisting of.