JP4841825B2 - Polyaniline / β-1,3-glucan complex - Google Patents

Description

  The present invention relates to a technique for highly dispersing polyaniline, particularly emeraldine salt type polyaniline, which is a conductive polymer, in an aqueous medium and further imparting functionality such as biochemical molecular recognition ability.

  Polyaniline is a black powdery polymer that is easily synthesized by oxidative chemical polymerization or electrochemical polymerization of aniline, and has long been known as aniline black. Another mechanism is that polyaniline has excellent chemical stability and the addition of protons to nitrogen atoms, in addition to the fact that conductivity develops by redox of conjugated electron systems as in normal conducting polymers. The research is actively conducted because of the involvement of.

  Polyaniline can take two structures, an amine having a reduced nitrogen atom and an oxidized imine, and has a very complicated molecular structure due to the protonation of the nitrogen atom. After the oxidative polymerization of aniline, an emeraldine salt type polyaniline is obtained. When this is alkali-treated, it becomes an emeraldine base type. Further, when this is chemically or electrochemically reduced, it is converted to leuco emeraldine base type polyaniline (see FIG. 1). Of these four types of polyaniline, only emeraldine salt type polyaniline exhibits conductivity.

  Although the properties unique to polyaniline, which are not found in such other conductive polymers, have been attracting attention for a long time, it cannot be said that the unique properties have been sufficiently exploited to date. This is probably because polyaniline has low solubility in not only water but also organic solvents. Furthermore, when considering use as a nanomaterial, it is necessary to pay attention to the function of each polyaniline fiber, and thus it is necessary to disperse and disperse one fiber in isolation. However, it is hard to say that research is in progress from this point of view.

So far, many researchers have independently developed a method for stably dissolving polyaniline in a solvent, particularly water. For example, there are a method of grafting a water-soluble polymer onto polyaniline, a method of blending a water-soluble molecule such as camphorsulfonic acid with polyaniline, and a method of reacting polyaniline with chlorosulfuric acid to convert it into a water-soluble derivative. If water-solubility can be imparted to polyaniline, it becomes possible to make polyaniline biocompatible, and application to new fields such as sensing elements targeting physiologically important nucleic acids and proteins is expected.
JP-A-10-92220 Patrick A. McCarthy, JianyuHuang, Sze-Cheng Yang, Hsing-Lin Wang, Langmuir, 18, 259-263 (2002) JP 10-60108 A

  However, even if polyaniline solubilization can be achieved by the method described in the above-mentioned document, problems such as changes in properties due to derivatization and labor and cost required for the reaction remain. It is considered that it is extremely difficult to impart a recognition site necessary for the sensing element to polyaniline simultaneously with water solubilization by the conventional method. Moreover, the product obtained by the conventional method does not form a specific structure at the nano level. When targeting a biological substance, it is necessary to detect a very small amount of sample with high sensitivity, and thus it is necessary to disperse and functionalize the polyaniline fiber at the nano level. Therefore, further microfabrication is required to use such a material as a nanomaterial.

  An object of the present invention is to provide a conductive polyaniline that is highly dispersed in water while maintaining the chemical structure of the polyaniline, achieves solubilization and fiberization, and is further provided with biochemical recognition ability as necessary. It is intended to provide a technique for preparing the above.

The present inventors have found that the above object can be achieved by complexing β-1,3-glucan of natural polysaccharide and polyaniline.
Thus, the present invention provides a water-dispersible complex comprising polyaniline (particularly emeraldine salt type polyaniline) and β-1,3-glucan. Furthermore, according to the present invention, there is provided a method for producing a water-dispersible complex comprising such polyaniline and β-1,3-glucan, which contains a single-chain β-1,3-glucan. By mixing an aprotic polar solvent solution or an aqueous alkaline solution with an aprotic polar solvent solution containing emeraldine base type polyaniline, adding water under stirring and further incubating, emeraldine base type polyaniline and β A method is provided comprising the step of forming a complex consisting of -1,3-glucan.

  According to the present invention, commercially available polyaniline can be dissolved and highly dispersed in water by a simple physical operation, and conductive polyaniline imparted with biochemical recognition ability can be provided. It is expected to promote use in the biotechnology field.

  As is well known, the β-1,3-glucan used in the present invention is a polysaccharide in which glucose is bound by a β1 → 3 glucooxide bond. Β-1,3-glucan produced as a natural polysaccharide forms a triple helical structure in water. The hydrophobic space in the central portion is considered to have a diameter of 1 nm or less, and even if mixed with polyaniline, it is difficult to put the polyaniline fiber into the space as it is.

  However, according to the present invention, a solution of β-1,3-glucan previously dissolved in an aprotic polar solvent or an alkaline aqueous solution and dissociated into a single chain, and a polyaniline dissolved in an aprotic polar solvent, Are mixed and stirred while adding water, followed by incubation to easily obtain a complex composed of polyaniline and β-1,3-glucan. It is known that when β-1,3-glucan dissociated into a single strand state is put into neutral water, it tries to sprinkle back into a triple helix. It is understood that β-1,3-glucan is wound and wrapped around the polymer (polyaniline), and a complex of polyaniline and β-1,3-glucan is formed.

  A particularly suitable example for use as an aprotic polar solvent that dissolves β-1,3-glucan and / or an aprotic polar solvent that dissolves polyaniline is dimethyl sulfoxide (DMSO). An example of a suitable alkaline aqueous solution in which β-1,3-glucan is dissolved and dissociated into a single chain is an aqueous caustic soda solution. In general, the incubation is preferably performed at a temperature of about 50 ° C. for 2 days.

  As shown also in the examples described later, the polyaniline / β-1,3-glucan complex of the present invention is composed of one-dimensionally oriented polyaniline (fibrous polyaniline) and β-1,3-glucan in the hydrophobic space. It is understood that the structure is taken up and coated. FIG. 2 schematically shows the structure of such a polyaniline / β-1,3-glucan complex of the present invention.

  The polyaniline / β-1,3-glucan of the present invention is extremely excellent in water dispersibility (solubility in water) without causing a chemical change in polyaniline. That is, according to the method of the present invention, by mixing both components of polyaniline and β-1,3-glucan under specific conditions, water-solubilization of polyaniline and nano-level refinement (one-dimensional orientation) can be achieved. Can be easily achieved.

  When polysaccharides other than β-1,3-glucan are used, polyaniline is slightly soluble in amylose under low concentration conditions, but the stability as a solution and the dispersibility in fiber form are almost unacceptable. Moreover, the solubility is not recognized about starch, dextrin, and pullulan (refer the below-mentioned Example).

  The complex of the present invention is generally a complex of emeraldine base type polyaniline and β-1,3-glucan by applying the above-described method using polyaniline commercially available as emeraldine base type as a starting material. It is also possible to obtain an emeraldine salt type polyaniline / β-1,3-glucan complex by converting the emeraldine base type polyaniline into an emeraldine salt type polyaniline by acidifying the complex in an aqueous medium. it can. For example, when a small amount of hydrochloric acid is added, protons are doped and the solution is changed to an emeraldine salt type polyaniline aqueous solution which is a conductive polymer. It has also been confirmed that such proton doping does not affect the morphology of the composite. The emeraldine salt type or emeraldine base type polyaniline constituting these complexes, as is well known in the art, is subjected to leuco emeraldine salt type or leuco emerald by being subjected to appropriate reducing conditions. Din base type polyaniline can be changed.

  The β-1,3-glucan that can be used in the present invention includes various β-1,3-glucan compounds obtained as natural polysaccharides. In particular, about 30 to 50% of the side chain is glucose-substituted. Schizophyllan, scleroglucan and lentinan are preferably used. Even β-1,3-glucan having no glucose substituent in the side chain can have a relatively low molecular weight and high solubility, for example, curdlan.

Furthermore, it is also possible to introduce various functional functional groups into the side chain of β-1,3-glucan in a regioselective manner, which makes it possible for polyaniline to function via β-1,3-glucan. Grant is also expected. That is, the obtained complex has the conductivity or redox activity of polyaniline and the biocompatibility of polysaccharide at the same time, and the application to unprecedented biomaterials, especially biosensors, is also open.
The characteristics of the present invention will be described more specifically in the following examples, but the present invention is not limited to these examples.

Preparation of low molecular weight curdlan Add 3.0 g of curdlan with molecular weight of 1.2 million (Wako Pure Chemical Industries, Ltd., CAS Registry Number 9051-97-2) to 50 ml of dry DMF (dimethylformamide) and heat the solution to 50 ° C And dissolved overnight. To this, 0.3 g of paratoluenesulfonic acid was added to start the hydrolysis reaction. While heating and stirring at 50 ° C., the progress of the reaction was followed by gel permeation liquid chromatography (GPC), and 15 ml of the reaction solution was fractionated when hydrolyzed to the target molecular weight. 200 ml of methanol was added to the separated reaction solution, and purification was performed by reprecipitation. The resulting white fibrous precipitate was dried to obtain a low molecular weight curdlan.
Pullulan reference material (Pullulan standard)
Standard, SHOWA DENKO KK) as a standard and measured by the GPC method. Measurement conditions are liquid feeding system: JASCO, PU-1580, column oven: JASCO, CO-2060, detector: JASCO, RI-2031, column: TOSOH TSK-gel α-4000, moving layer: DMF, Flow rate: 0.6ml / min, temperature: 40 ° C. As a result, it was found that Mn = 19,000, Mw = 33,300, Mw / Mn = 1.75, and it was confirmed that the product was soluble in water. It was done.

Dissolution of polyaniline by curdlan The mixing was performed in the following procedure at the ratio shown in Table 1. First, the DMSO solution of curdlan obtained in Example 1 (0.5 g / dl) and the DMSO solution (0.1 g / dl) of polyaniline (purchased from Aldrich, emeraldine base type, Mw = 10,000) were mixed and homogeneously mixed. It was set as the solution. Distilled water was added to this solution little by little with stirring so as to keep the solution homogeneous. Finally, 1.9 ml of water was added to make water: DMSO = 95: 5 (v / v) (Vw (volume ratio of water) = 95%). This solution was incubated at 50 ° C. for 2 days. The obtained solution was transferred to a centrifuge tube and centrifuged (7000 rpm) for 1 hour to precipitate the curdlan-polyaniline complex, and the unreacted curdlan and the supernatant containing polyaniline were removed. The precipitate was dispersed in 2 ml of water and further centrifuged (7000 rpm) for 1 hour. This operation was repeated three times to finally obtain an aqueous solution of curdlan-polyaniline complex. This solution showed a blue color peculiar to emeraldine base type polyaniline, indicating that polyaniline was stably dispersed in water.

Preparation of schizophyllan schizophyllan having a triple helix structure was produced according to a standard method described in the literature. That is, ATCC (American Type
Schizophyllum from the Culture Collection
commune. Fries (ATCC 44200) was cultured in a minimal medium for 7 days, and the supernatant obtained by centrifuging cell components and insoluble residues was sonicated to obtain a molecular weight of 450,000 (single strand) 150,000) of triple helix schizophyllan.
Gregory G. Martin, Michael F. Richardson, Gordon C. Cannon and Charles L. McCormick, Am. Chem. Soc. Polymer Prepr. 38 (1), 253-254 (1997) Kengo Tabata, Wataru Ito, Takemasa Kojima, Shozo Kawabata and Akira Misaki, CarbohydrateResearch, 89, 121-135 (1981)

Dissolution of polyaniline with schizophyllan DMSO solution (2.0 g / dl) of schizophyllan (designated SPG in the drawing) obtained in Example 3 and DMSO solution (0.4 g / dl) of polyaniline (designated PANI in the drawing) ) Were mixed and dispersed uniformly. Distilled water was added to this solution little by little with stirring. Finally, 1.9 ml of water was added to make water: DMSO = 95: 5 (v / v) (Vw = 95%). This solution was incubated at 50 ° C. for 2 days. The obtained liquid was transferred to a centrifuge tube, and centrifuged (7000 rpm) for 1 hour to precipitate schizophylan / polyaniline complex, and the supernatant containing unreacted schizophyllan and polyaniline was removed. The precipitate was dispersed in 2 ml of water and further centrifuged (7000 rpm) for 1 hour. This operation was repeated three times, and finally a complex aqueous solution of schizophyllan and polyaniline was obtained. Table 2 shows the mixing ratio and the result of final dispersibility evaluation.

Comparative Example 1
Dissolution of polyaniline with various polysaccharides Dispersed under the same conditions as in Example 4 using amylose and starch which are polysaccharides other than β-1,3-glucan. The evaluation results of the mixing ratio and dispersibility are shown in Table 3. As a result, when these non-β-1,3-glucan polysaccharides were used, precipitates were formed, and polyaniline could not be dissolved in water under these conditions. When the sample concentration before mixing was reduced to 1/4 (under the conditions of Example 2), only complex formation and dissolution in water were observed for amylose (160,000). Triple chain schizophyllan did not show any solubility even at low concentrations.

And Comparative Example 2
Microscopic Observation of Polysaccharide-Polyaniline Complex Properties of the complex using schizophyllan (Example 4) and amylose (Comparative Example 1) obtained under the conditions in Table 2 were compared by AFM and TEM observation. As a result of AFM observation, when amylose was used, almost no fiber was observed, and it was agglomerated, but the one using schizophyllan was observed to have a fiber shape. This result also corresponds to the result of TEM observation (Schizophyllan: FIG. 3a, b amylose: FIG. 3c). From these results, it can be concluded that schizophyllan has incorporated polyaniline into a fiber form in its space and is solubilized.

And Comparative Example 3
Circular dichroism (CD) spectrum of polysaccharide-polyaniline complex It was confirmed by using CD spectrum that polyaniline and schizophyllan formed a complex. Although only the polyaniline used has no asymmetric structure, CD is inactive, but it is thought that polysaccharide-derived CD is induced in polyaniline by interacting with schizophyllan. FIG. 4 shows the CD spectrum of the complex aqueous solution obtained under the conditions shown in Table 2. From FIG. 4, CD was observed in the absorption region of polyaniline. This indicates that polyaniline and schizophyllan interact at the molecular level to form a complex.

And Comparative Example 4
UV-Visible Spectrum Observation of Polysaccharide-Polyaniline Complex Property comparison of the complex using schizophyllan (Example 4) and amylose (Comparative Example 1) obtained under the conditions in Table 2 was performed by UV spectrum measurement. . In the spectrum shown in FIG. 5, the peak at 600 nm is attributed to the peak derived from the quinoid type structure in the emeraldine structure, and the peak at 300 nm is attributed to the peak derived from the benzonoid type. From this figure, it is shown that polyaniline is stably dissolved in water by polysaccharide without causing any chemical change.
Although it seems that there is no difference in the spectra of amylose and schizophyllan, it has been confirmed that the two peaks derived from schizophyllan-polyaniline are shifted by a short wavelength of about 5 nm, respectively, compared to that of amylose-polyaniline. Similarly, in the sample using schizophyllan, a monochromatic effect in which the peak intensity decreases can also be confirmed. These results indicate that the polyaniline fibers are closely aligned in the pores of schizophyllan. That is, it is considered that schizophyllan has taken polyaniline into its hydrophobic space more strongly than amylose.
AtushiWatanabe, Toyoki Kunitake, J. Colloid Interface Sci., 145, 90-98 (1991)

And Comparative Example 5
Stability Evaluation of Polysaccharide-Polyaniline Complex by pH Change The stability of the complex by pH change when using schizophyllan (Example 4) and amylose (Comparative Example 1) obtained under the conditions shown in Table 2 was examined. As described above, the most important structure of polyaniline is an emeraldine salt type polymer, and it is known that it can be easily obtained by doping an emeraldine base with a protonic acid. Therefore, an attempt was made to dope the obtained composite with hydrochloric acid. Samples were prepared by adding 5 μl of hydrochloric acid to 2.0 ml of each aqueous solution. The UV spectrum immediately after doping with hydrochloric acid is shown in FIG. 6a, and the spectrum after 24 hours is shown in FIG. 6b. The color of the solution changed from blue to green by hydrochloric acid dope. In the UV spectrum, with the disappearance of the peak at 600 nm, a large, broad peak appeared in the long wavelength region. This is a peak derived from a cation radical (polaron) generated by doping, and it was shown that isomerization by doping of the complex is possible as in the case of ordinary polyaniline. The schizophyllan-polyaniline complex was stable in the acidic region, but it was confirmed that the amylose-polyaniline complex produced a green precipitate several hours after the addition of hydrochloric acid.
The UV spectrum after 24 hours shows almost no change in the waveform of the schizophyllan-polyaniline complex compared with that immediately after the addition of hydrochloric acid, whereas the amylose-polyaniline complex has a complete peak derived from polyaniline in the solution. Had disappeared. The reason why the schizophyllan-polyaniline complex retains high stability even under acidic conditions is that polyaniline is incorporated into the hydrophobic space inside the schizophyllan, and is due to the polyaniline being completely covered with schizophyllan. it is conceivable that. In fact, it became clear as a result of observation by TEM (Transmission Electron Microscope) that the composite remained in a fiber-like form even after doping with hydrochloric acid (FIG. 7). On the other hand, as shown by AFM and TEM, amylose does not take such a solubilized form.

Functional addition to polyaniline A great merit of wrapping polyaniline with β-1,3-glucan such as schizophyllan is that various functions can be given to polyaniline by chemically modified schizophyllan. Therefore, in order to evaluate the functionalization and biocompatibility of polyaniline, it was decided to solubilize polyaniline with mannose-modified schizophyllan and to evaluate the concanavalin A (ConA) affinity of the resulting complex. ConA is a type of sugar-binding protein that interacts specifically with mannose. If the binding of ConA to the complex can be confirmed, polyaniline can be coated with polyaniline in the same way that chemically modified schizophyllan can be coated. It can be confirmed that affinity can be imparted. In order to visually capture this phenomenon, samples were prepared using ConA (FITC-ConA) labeled with a fluorescent dye and evaluated with a confocal laser microscope. Sample preparation conditions were the same as in the case of unmodified schizophyllan.

The results of observation with a confocal laser microscope are shown in FIG. FIG. 8a is a fluorescent image showing only fluorescently labeled ConA. On the other hand, FIG. 8b is a transmitted light image, and a composite image appears. It can be seen from FIG. 8c that these two images are completely coincident. These results indicated that ConA was bound to the surface of the complex. In addition, in FIG. 8, in order to make an understanding easy, the figure drawn by tracing the image of each micrograph by hand is also shown.
From the above results, it was shown that protein recognition ability can be imparted to polyaniline via chemically modified schizophyllan. In addition, since protein (ConA) is bound to the complex surface without losing its sugar-binding ability, biocompatibility can be imparted by coating polyaniline with schizophyllan. If these results are used, various functional groups can be introduced into β-1,3-glucan such as schizophyllan, and if combined with polyaniline, various functions can be provided by simply mixing the two.
As shown in the above examples, according to the present invention, polyaniline water-solubilization, one-dimensional orientation (fibrosis), and functionalization can be performed simultaneously and simply. It is thought to contribute to promoting the application as a nanodevice.

  The polyaniline / β-1,3-glucan complex obtained by the present invention contributes to the development of new uses of polyaniline. In particular, the complex containing water-soluble conductive polyaniline is composed of a transparent electrode, a photoelectric conversion element, an organic Use in fields such as electroluminescence elements, catalysts, nonlinear optical materials, biochemical sensor elements and the like is expected.

The isomers of polyaniline to which the present invention is applied and their interconversion relationships are shown. The conceptual diagram of the β-1,3-glucan-polyaniline complex of the present invention is shown. The TEM image (Example 5 and Comparative Example 2) of a schizophyllan-polyaniline complex and an amylose-polyaniline complex is shown. The CD spectrum (Example 6 and Comparative Example 3) of a schizophyllan-polyaniline complex is shown. The UV spectrum (Example 7 and Comparative Example 4) of a schizophyllan-polyaniline complex and an amylose-polyaniline complex is shown. The stability (Example 8 and Comparative Example 5) of the UV spectrum by the pH change of the schizophyllan-polyaniline complex and the amylose-polyaniline complex is shown. The TEM image (Example 8) of the schizophyllan-polyaniline complex after hydrochloric acid dope is shown. It is a confocal laser microscope image (Example 9) which shows that a mannose modification schizophyllan-polyaniline complex couple | bonds with ConA.

Claims (2)

  A water-dispersible complex comprising polyaniline and β-1,3-glucan, wherein the polyaniline is an emeraldine salt type polyaniline and the β-1,3-glucan is a curdlan. Complex. A method for producing a water-dispersible composite comprising emeraldine salt polyaniline and curdlan,
Mixing an aprotic polar solvent solution or alkaline aqueous solution containing a single-stranded curdlan with an aprotic polar solvent solution containing emeraldine base type polyaniline, adding water with stirring, and further incubating Forming a complex comprising emeraldine base type polyaniline and curdlan, and emeraldine by acidifying the resulting complex comprising emeraldine base type polyaniline and curdlan in an aqueous medium. A method comprising the step of converting a base type polyaniline into an emeraldine salt type polyaniline.