JP2014212774A - Fluorescent dye - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cheap fluorescent dye using biomass as a raw material being cheap and constantly available in a large mass, the dye fluorescing stably for a long period of time without flickering.SOLUTION: Lignin extracted from biomass by using an aqueous solution of acetic acid is used as the main component of the fluorescent dye of the invention. If desired, a mineral acid such as sulfuric acid may be used with the acetic acid aqueous solution for extraction. Since the main component lignin is a stable material, irradiation of excitation light does not cause color fading, and fluorescent light does not flicker. Further, the lignin can be stored at room temperature making its management easy. Also, because of its stability, the fluorescent dye may be micronized to enable its introduction into living cells. The fluorescent dye of the invention strongly fluoresces when irradiated with an excitation light of a wavelength around from 488 nm to 566 nm, so that it is useful as the fluorescent dye for cell observation under a fluorescence microscope.

Description

  The present invention relates to a fluorescent dye. More specifically, the present invention relates to a fluorescent dye containing a specific lignin as a main component.

  In tissue regeneration and pathological diagnosis, a technique for evaluating the distribution and dynamics of proteins expressed inside and outside cells is important. One of the requirements in protein evaluation is to bind a marker to the protein of interest. At present, a method using a fluorescent dye as a marker is common. Specifically, when evaluating the protein distribution, after binding the fluorescent dye-labeled antibody to the target protein of the cell, the wavelengths 405, 488 conventionally used as excitation light using a fluorescence microscope, Visible light in the vicinity of 566 and 633 nm is irradiated to the fluorescent dye, and the generated fluorescence is observed. In the above-described method, it is very important that the fluorescent dye generates fluorescence with excitation light having a wavelength of about 405 nm to 688 nm and that the fluorescence is stable.

  When evaluating the dynamics of a protein in a living cell, after introducing a fluorescent dye-labeled antibody into the living cell, there is a method for evaluating the locus of the target protein bound to the fluorescent dye using a fluorescence microscope. Even in this method, in order to obtain a good evaluation result, a stable fluorescent property is required for the fluorescent dye. In addition, since it is also important in the above-described evaluation to examine in detail the amount of fluorescent dye to be introduced into cells, a large amount of fluorescent dye is used in preliminary experiments. However, since the fluorescent dye is expensive, its use is a burden on the user.

  As a fluorescent dye used for cell observation in a fluorescence microscope, there is a fluorescent dye manufactured by chemical synthesis. Examples of such fluorescent dyes include substances in which rhodamine or fluorescein is modified with isothiocyanate so as to bind to proteins (Patent Documents 1 and 2). However, when these fluorescent dyes are irradiated with excitation light for a long time, fluorescence fading occurs, and it is difficult to observe stably for a long time with a fluorescence microscope. In particular, in a protein with a small expression level, there is a possibility that the expression of the protein cannot be confirmed due to fluorescence fading. Further, Patent Documents 3 and 4 describe methods using quantum dots as fluorescent dyes. However, since quantum dots may cause flashing of light, it is not easy to obtain a stable result in an experiment for evaluating the locus of fluorescence. Another problem is that conventional fluorescent dyes are expensive and therefore limited in their use. The factor that makes fluorescent dyes expensive is that the above-described step of adding isothiocyanate is required. Furthermore, conventional fluorescent dyes must be stored at a low temperature, which places a heavy burden on the user in management.

Japanese Patent No. 4603109 Japanese Patent Application Laid-Open No. 2007828846 US Pat. No. 6,194,213 US Pat. No. 6,306,610

  Accordingly, an object of the present invention is to provide an inexpensive fluorescent dye that is stably generated for a long time without blinking fluorescence.

  In order to achieve the above object of the present invention, the present inventor paid attention to biomass. Biomass components are known to generate fluorescence when irradiated with specific excitation light. One example is lignin. Lignin is known to generate fluorescence when irradiated with ultraviolet rays. As one of methods for evaluating lignin in plants, a method has been reported in which ultraviolet rays are irradiated to plant sections and the presence or absence of lignin is evaluated from the fluorescence luminance. As a method for recovering lignin from biomass, there is an atmospheric pressure acetic acid pulping method (Holzforschung, 49 (1995) pp.343-350). However, as the application field of lignin recovered by the atmospheric pressure acetic acid pulping method, conventionally, only carbon fiber, a reinforcing agent for molded articles, and the like are considered. Moreover, the utilization method in the case of refinement | miniaturization is not examined.

As a result of intensive studies, the present inventor has surprisingly found that lignin extracted with acetic acid is excited by visible light and emits fluorescence, and has reached the present invention. It has not been known so far that lignin is excited by visible light, particularly visible light in the vicinity of 488 to 566 nm and generates stable fluorescence.
That is, the present invention is as follows.

1. A fluorescent dye that contains lignin extracted from biomass using an aqueous acetic acid solution as a main component and emits fluorescence with excitation light having a wavelength of about 488 to 566 nm.
2. The fluorescent dye according to 1 above, wherein the biomass is a residue obtained by partially decomposing holocellulose by hydrolysis.
3. The fluorescent dye according to 1 or 2 above, wherein lignin is extracted from biomass using an aqueous acetic acid solution and a mineral acid.
4). The fluorescent dye according to 1, 2 or 3, wherein the melting point and thermal decomposition temperature of lignin are 100 ° C or higher.
5. 5. The fluorescent dye according to any one of 1 to 4 above, wherein the average particle size of lignin is in the vicinity of 10 to 100 nm.
6). 6. The fluorescent dye according to any one of 1 to 5 above, which is used for labeling a fluorescent dye of a protein.
7). 6. The fluorescent dye according to 6 above, wherein lignin and an antibody are combined.

The fluorescent dye of the present invention is characterized in that it emits fluorescence when irradiated with excitation light having a wavelength of about 488 to 566 nm, and its fluorescence intensity is higher than that of commercially available lignin. In addition, since lignin, which is the main component of the fluorescent dye of the present invention, is a stable substance, fading due to irradiation with excitation light does not occur and light does not blink. Furthermore, management is simple because it can be stored at room temperature. In addition, since it is stable, it can be miniaturized so that it can be introduced into living cells.
Furthermore, since the raw material of the fluorescent dye of the present invention is biomass, it can be stably obtained in a large amount at a low cost. In addition, since lignin has the property of adsorbing proteins, when it is used as a fluorescent dye for cell observation, chemical modification for binding to the target protein is not necessary. From the above, it becomes possible to produce an inexpensive fluorescent dye.

It is a flowchart of the manufacturing method and the usage method in the fluorescent dye particle of this invention. It is an image which shows the test result about the fluorescence characteristic of lignin collect | recovered with the acetic acid of this invention. It is an image which shows the test result about the fluorescence characteristic of a clason lignin. It is an image which shows the test result about the fluorescence characteristic of a cellulose acetate.

The present invention is described in detail below.
The fluorescent dye is preferably a substance having a thermal decomposition temperature of 100 ° C. or higher when the weight reduction rate is defined as 5%. The reason is that if the temperature is 100 ° C. or lower, there is a possibility of thermal decomposition in the process of miniaturization. It is also preferable for the same reason that the substance does not have a melting point at 100 ° C. or lower. Furthermore, it is required to be a substance having the property of adsorbing to proteins such as cellulase. Lignin is a preferred material in this respect.

  The fluorescent dye of the present invention is mainly composed of lignin extracted from biomass using an acetic acid aqueous solution and, if necessary, a mineral acid. This fluorescent dye emits strong fluorescence when irradiated with excitation light having a wavelength of about 488 nm to about 566 nm generally used in a fluorescence microscope.

  Biomass used as a raw material of the present invention is generally defined as a renewable, organic organic resource excluding fossil resources. More specifically, there are herbaceous biomass and woody biomass. In the herbaceous biomass, for example, rice straw, straw, bamboo, rice husk, wheat pattern and the like can be used. In woody biomass, for example, coniferous trees such as cedar, pine, and cypress can be used. Furthermore, even if it is a residue discharged | emitted when the holocellulose of the said biomass is hydrolyzed by hydrothermal treatment, an enzyme saccharification process, etc., if it is a residue containing a lignin, it can be used for this invention. Further, even biomass that has been subjected to mechanical treatment such as pulverization can be used.

The method for producing the fluorescent dye of the present invention will be described with reference to FIG.
(Step S101)
In the production of a fluorescent dye, lignin is first extracted from biomass using an aqueous acetic acid solution. The concentration of the acetic acid aqueous solution is not particularly limited, but is usually 40 to 90% by volume, preferably 80 to 90% by volume. Extraction can be smoothly carried out by adding a mineral acid such as hydrochloric acid or sulfuric acid to an aqueous acetic acid solution, but this is not always necessary when extraction in a short time is not required. When adding a mineral acid, the density | concentration is 0.1-1.0 volume% normally, Preferably it is 0.1-0.5 volume%.
Extraction is possible even at room temperature, but it is preferable because lignin can be extracted more quickly by heating. Usually, the extraction pressure is atmospheric pressure. The extraction time is usually 12 to 48 hours, preferably 24 to 48 hours when no mineral acid is used, and usually 1 to 12 hours, preferably 3 to 6 hours when used.

Subsequently, the extract of lignin and the residue are separated by a known filtration device or the like, and the extract is recovered. The recovered extract is concentrated by vacuum concentration or the like. In that case, it is preferable to heat at about 100-118 degreeC. After adding distilled water to the concentrated liquid, the precipitated lignin is separated and recovered. The recovery can be performed with a known filtration device or the like. The recovered lignin is dried by drying under reduced pressure or the like.
The lignin thus obtained is a powder. Further, if the particle size is 100 nm or less before drying, it can be used as it is as the fluorescent dye of the present invention.

(Step S102)
The lignin obtained in step S101 as described above can be used as a fluorescent dye if it is sufficiently fine, but can be further refined and used as necessary. For example, a pulverization process is used to refine lignin. When the pulverization is performed, a known wet rotating ball mill or bead mill can be used. The refined lignin is dried by a known method. The average particle size of the lignin thus obtained is preferably 10 to 100 nm, more preferably 10 to 20 nm.

(Step S103)
When the fluorescent dye of the present invention is used for protein detection, it is preferably a complex bound to an antibody. As an antibody (hereinafter referred to as a primary antibody), one that binds to the target protein is selected. The complex is formed by mixing lignin and the primary antibody in distilled water or phosphate buffer. In order to strengthen the binding between the lignin and the primary antibody, the lignin is dried after immersing the lignin in the amino group of the primary antibody and the silane coupling agent or glutaraldehyde. Thereafter, the amino group of the primary antibody and lignin are bound in a solution such as distilled water or phosphate buffer.

When introducing into living cells in which fine holes are formed in the cell membrane, the cell membrane is treated using a surfactant or electroporation. Further, the above-described complex may be directly injected into the cell by a microinjection method.
When the complex described above is introduced into cells fixed with formalin, paraformaldehyde phosphate buffer, or the like, the cell membrane is first treated with a surfactant. Thereafter, the above-described complex is introduced.

After the above steps, the lignin-bound antibody binds to the target protein of the cell. Thereafter, the intracellular protein can be observed by irradiating visible light having a wavelength of 488 to 566 nm with a fluorescence microscope to generate fluorescence and observing the intracellular fluorescent dye.
In addition, the above observation becomes clearer by adding Bovine serum albumin (BSA) before introducing the lignin-bound antibody into the cells.
When observing a protein expressed outside the cell membrane, the target protein is labeled by adding lignin bound to the primary antibody to living cells in culture and cells fixed with formalin or paraformaldehyde. Thereafter, the protein with a cell membrane can be observed by observing with a fluorescence microscope.

Hereinafter, the present invention will be described with reference to Examples and Comparative Examples.
Example 1
[Fluorescence characteristics of lignin extracted with acetic acid and mineral acid]
Cellulase was added to a cedar fine powder refined to an average particle size of 25 μm by a vibration pulverizer in an acetic acid buffer having a pH of 5.5. Then, it heated at 50 degreeC for 24 hours. This is the biomass saccharification treatment. Thereafter, the residue was collected using a filtration filter.
The procedure for recovering lignin from the residue is as follows. 400 mL of an 80% by volume acetic acid aqueous solution was poured into 40 g of the residue contained in the container, and then 0.4 mL of hydrochloric acid (11.7 mM) was added. Thereafter, the lignin was digested by heating at 130 ° C. for 6 hours. After collecting only the liquid extracted from the lignin using a suction filtration device, the lignin was precipitated by concentration under reduced pressure and addition of distilled water. Then, lignin was collect | recovered by drying with a freeze dryer. The recovered lignin had a particle size of about 30-60 μm.
The particle size was measured by appropriately using a light scattering method and a scanning electron microscope (SEM).

  FIG. 2 shows the results for the fluorescence property test of lignin extracted with acetic acid. In the test, lignin was sandwiched between a slide glass and a cover glass and observed using a confocal laser microscope. The wavelengths of the irradiated excitation light are 405, 488, 566, and 633 nm. When lignin was irradiated with excitation light (laser intensity 40%) having wavelengths of 488, 566, and 633 nm, fluorescence was observed. In particular, the fluorescence intensity was high at wavelengths of 488 and 566 nm. Further, it was possible to confirm fluorescence even at the lowest laser intensity of 5%. The laser intensity of 5% is a laser intensity used in general cell observation. Therefore, the lignin recovered using acetic acid can be sufficiently used as a raw material for the fluorescent dye.

Example 2
[Fluorescence characteristics of lignin extracted with acetic acid]
Cellulase was added to cedar fine powder refined to an average particle size of 25 μm by a vibration pulverizer in an acetic acid buffer having a pH of 5.5. Then, it heated at 50 degreeC for 24 hours. Thereafter, the residue was collected using a filtration filter.
The procedure for recovering lignin from the residue is as follows. 400 mL of 80% by volume acetic acid aqueous solution was poured into 40 g of the residue contained in the container, and the lignin was digested by heating at 130 ° C. for 6 hours. After collecting only the liquid extracted from the lignin using a suction filtration device, the lignin was precipitated by concentration under reduced pressure and addition of distilled water. Then, lignin was collect | recovered by drying with a freeze dryer. The characteristics of the collected lignin were the same as in Example 1.

Comparative Example 1
[Fluorescence characteristics of clason lignin]
The result about the fluorescence characteristic test of commercially available clason lignin is shown in FIG. The test method is the same as in Example 1. It was confirmed that clason lignin generates fluorescence when the wavelength of the excitation light is 405 and 566 nm (laser intensity 40%). However, compared with Example 1, it turned out that the fluorescence intensity in excitation light of 566 nm is low.

Comparative Example 2
Fluorescence characteristics of cellulose acetate The results of the fluorescence characteristics test of commercially available cellulose acetate are shown in FIG. The test method is the same as in Example 1. It was confirmed that cellulose acetate did not produce fluorescence in excitation light with wavelengths of 488, 566, and 633 nm. From this, it was found that the fluorescence of Example 1 was caused by lignin modified with acetic acid, not acetic acid.

  Since the fluorescent dye of the present invention generates strong fluorescence when irradiated with excitation light having a wavelength of about 488 to 566 nm, it is useful as a fluorescent dye for cell observation in a fluorescence microscope.

Claims (7)

  A fluorescent dye that contains lignin extracted from biomass using an aqueous acetic acid solution as a main component and emits fluorescence with excitation light having a wavelength of about 488 to 566 nm.   2. The fluorescent dye according to claim 1, wherein the biomass is obtained by partially decomposing holocellulose by hydrolysis.   The fluorescent dye according to claim 1 or 2, wherein the lignin is extracted from biomass using an acetic acid aqueous solution and a mineral acid.   The fluorescent dye according to claim 1, 2 or 3, wherein the melting point and thermal decomposition temperature of lignin are 100 ° C or higher.   The fluorescent dye according to any one of claims 1 to 4, wherein the average particle diameter of lignin is around 10 to 100 nm.   The fluorescent dye according to any one of claims 1 to 5, which is used for labeling a fluorescent dye of a protein. The fluorescent dye according to claim 6, wherein lignin and an antibody are combined.