JP7477869B2 - Composite particles for recovering electric bacteria, method for producing composite particles for recovering electric bacteria, and method for recovering electric bacteria - Google Patents

Description

The present invention relates to composite particles for recovering electric bacteria, a method for manufacturing composite particles for recovering electric bacteria, and a method for recovering electric bacteria.

A method is known for recovering cells from a liquid medium containing impurities using magnetic beads that have an affinity for cells. Patent Document 1 describes a nucleic acid extraction method comprising adding magnetic beads to a specimen containing cells, capturing the cells on the magnetic beads, separating the cells captured by the magnetic beads from the specimen using a magnetic force, and extracting nucleic acid from the cells using the magnetic beads.

JP 2007-89563 A

In recent years, research on "electrobacteria" that have electron transfer enzymes as membrane proteins has been progressing. In this context, a method for easily isolating and recovering electrobacteria from bacterial flora is required.
The method of Patent Document 1 makes it possible to recover specific cells by fixing antibodies or other substances that have affinity for desired cells to the surface of magnetic beads, but it is difficult to easily recover, for example, unknown electric bacteria or a wide variety of "electric bacteria" all at once.

Therefore, an objective of the present invention is to provide composite particles for recovering electric bacteria that can be used to recover electric bacteria in a simpler manner. Another objective of the present invention is to provide a method for producing composite particles for recovering electric bacteria, and a method for recovering electric bacteria.

As a result of extensive research into achieving the above object, the inventors have discovered that the above object can be achieved by the following configuration.

[1] A composite particle for electrochemical bacterial recovery, comprising: a magnetic nanoparticle; an organic compound bound to the surface of the magnetic nanoparticle; and osmium tetroxide bound to the organic compound.
[2] The organic compound has a hydroxyl group and an amino group, and is bonded to the magnetic nanoparticles via the hydroxyl group and to the osmium tetroxide via the amino group. A composite particle for recovering electric bacteria described in [1].
[3] The composite particle for recovering electric bacteria described in [1] or [2], wherein the organic compound contains folic acid.
[4] A composite particle for recovering electric bacteria according to any one of [1] to [3], wherein the magnetic nanoparticles include Fe ₃ O ₄ nanoparticles.
[5] A method for producing composite particles for recovering electric bacteria, comprising: reacting _{Fe3O4 nanoparticles} with _folic acid in a solvent to obtain precursor particles in which folic acid is bound to the surface of the _Fe3O4 nanoparticles; and reacting the precursor particles with osmium tetroxide in a solvent to obtain composite particles in which the osmium tetroxide is bound to the folic acid.
[6] A method for recovering electric bacteria, comprising: preparing a solution containing electric bacteria, 3,3'-diaminobenzidine, and hydrogen peroxide, staining the electric bacteria, contacting the stained electric bacteria with a composite particle for recovering electric bacteria described in any one of [1] to [4] to form a complex between the stained electric bacteria and the composite particle, and recovering the complex.
[7] The method for recovering electric bacteria described in [6], further comprising washing the stained electric bacteria before contacting the stained electric bacteria with the composite particles.
[8] The method for recovering electrobacteria described in [6] or [7], wherein the recovery of the complex is carried out using a magnet.
[9] Composite particles in which _Fe3O4 nanoparticles and _osmium tetroxide are bound to each other via folic acid.
[10] The composite particle according to [9], wherein the bond is a coordinate bond.

According to the present invention, it is possible to provide composite particles for recovering electric bacteria. In addition, according to the present invention, it is also possible to provide a method for manufacturing composite particles for recovering electric bacteria, and a method for recovering electric bacteria.

The present invention will be described in detail below.
The following description of the components may be based on a representative embodiment of the present invention, but the present invention is not limited to such an embodiment.
In this specification, a numerical range expressed using "to" means a range that includes the numerical values before and after "to" as the lower and upper limits.

[Composite particles for electrobacterial recovery]
The composite particle for recovering electric bacteria according to the embodiment of the present invention (hereinafter simply referred to as "composite particle") is a composite particle having a magnetic nanoparticle, an organic compound bonded to the surface of the magnetic nanoparticle, and osmium tetroxide (VIII) bonded to the organic compound. The organic compound typically has a hydroxy group and an amino group, and the magnetic nanoparticle and the organic compound are preferably bonded via the hydroxy group, and more preferably are typically bonded by a coordinate bond with an oxygen atom derived from the hydroxy group. In addition, the osmium tetroxide and the organic compound are preferably bonded via the amino group, and more preferably are typically bonded by a coordinate bond with a nitrogen atom derived from the amino group.

In the course of the research into electrobacteria, the inventors have discovered that electrobacteria can be stained with a solution containing 3,3'-diaminobenzidine (DAB) and hydrogen peroxide. Generally, peroxidase is used as the target enzyme for DAB staining of cells, etc. On the other hand, in the case of electrobacteria, hydrogen peroxide is reduced without the aid of peroxidase, catalyzed by electron transfer enzymes present in the cell membrane, generating active oxygen species, which results in the production of DAB polymers that cover the cell membrane of the electrobacteria. The detailed procedure will be explained in the Examples, but electrobacteria are defined as bacteria that can be stained by the above method.

In this composite particle, osmium tetroxide is fixed to the surface of magnetic nanoparticles via an organic compound. Osmium tetroxide has the property of selectively binding to the -CH=CH- bond of DAB polymers. Therefore, it specifically binds to electric bacteria in a state in which DAB polymers have been produced to cover the cell membrane. Furthermore, since this composite particle contains magnetic nanoparticles, it is possible to easily collect only electric bacteria from the bacterial flora. Note that the above is a speculated mechanism, and even if the problem of the invention is solved by a mechanism other than the above, it is considered to be within the technical scope of the present invention.

(Magnetic Nanoparticles)
The magnetic nanoparticles may be a ferrite having a composition of M(II)Fe ₂ O ₄ , or a magnetic iron compound having a ferrite containing the same as a main component. Examples of M(II) include Fe ²⁺ , Co ²⁺ , Ni ²⁺ , Mn ²⁺ , Zn ²⁺ , Mg ²⁺ , and Cu ²⁺ , and may contain these elements alone or in combination. In particular, the magnetic nanoparticles preferably contain Fe ₃ O ₄ , and are preferably made of Fe ₃ O ₄ , in order to obtain composite particles having the effects of the present invention that are more excellent. The magnetic nanoparticles may be commercially available or may be synthesized using a known method.
The average particle size of the magnetic nanoparticles is not particularly limited, but is preferably 1 to 1,000 nm, more preferably 10 to 200 nm, and even more preferably 20 to 100 nm.

(Organic Compounds)
The organic compound binds to the surface of the magnetic nanoparticles and further functions to fix osmium tetroxide (VIII) to each other. The organic compound is not particularly limited as long as it can bind to both the magnetic nanoparticles and osmium tetroxide, but it is preferable that the organic compound has a hydroxy group for binding to the magnetic nanoparticles and an amino group for binding to the osmium tetroxide.

The method for producing the composite particles will be described later, but typically, it is preferable to deprotonate the hydroxyl group to increase its nucleophilicity toward the metal atom contained in the magnetic nanoparticle, thereby forming a coordinate bond with the metal atom via an oxygen atom.
In addition, the organic compound preferably forms a coordinate bond with osmium oxide via the nitrogen atom of the amino group.

The molecular weight of the organic compound is not particularly limited, but the molecular weight of the organic compound is preferably from 50 to 1,000, and more preferably from 100 to 500, in that composite particles having a more uniform particle size can be easily obtained.
Such compounds include, for example, cysteine, cystine, and folic acid, with folic acid being more preferred.

(Method for producing composite particles)
The method for producing the composite particles is not particularly limited, but in terms of more easily obtaining the composite particles, it is preferable that the method includes the steps of reacting _{Fe3O4 nanoparticles} with folic acid in a solvent to obtain precursor particles in which folic acid is bound to the surface of the _{Fe3O4 nanoparticles} (Step A) and reacting the precursor particles with osmium tetroxide (VIII) in a solvent to obtain composite particles in which osmium tetroxide is bound to folic acid (Step B).

The solvent used in step A is not particularly limited, but preferably contains water, more preferably consists of water. Typically, it is preferable to add folic acid to the _Fe3O4 nanoparticles dispersed in the solvent to obtain precursor particles bound to _{the Fe3O4 nanoparticles via} the hydroxyl groups of the folic acid.
In this case, the content of folic acid in the reaction solution containing _{Fe3O4 nanoparticles} , folic acid, and a solvent is not particularly limited, but it is generally preferable to adjust it so that there are 0.3 to 3.0 moles of folic acid per 1 mole of _{Fe3O4 nanoparticles} .
In general, the solid content of the reaction solution is preferably adjusted to 0.001 to 10% by mass.

The solvent may contain a pH adjuster other than the above. The pH adjuster is not particularly limited, but may be, for example, sodium hydroxide. By adjusting the reaction solution to be basic with the pH adjuster, the deprotonation of the hydroxyl group of folic acid is facilitated, and as a result, the binding between _{Fe3O4 nanoparticles} and folic acid is facilitated.
In general, the reaction temperature in step A is preferably 5 to 50° C., and the reaction time is preferably 1 to 24 hours.

The present method for producing composite particles preferably further includes a step of washing the precursor particles obtained in step A (precursor particle washing step) before carrying out step B. By removing unreacted folic acid in the precursor particle washing step, the yield of composite particles is further improved.
The washing method is not particularly limited, and may be a method of dispersing the obtained composite particles in a washing solvent (e.g., water) and recovering the solid content from the dispersion. Washing may be repeated several times.

The solvent used in step B is not particularly limited, and the same solvent as that used in step A can be used. By reacting the precursor particles with osmium tetroxide (VIII) in a solvent, composite particles in which osmium tetroxide (VIII) is bound via the amino groups of _folic acid fixed to the surface of _Fe3O4 nanoparticles are obtained.

The content of osmium tetroxide in the reaction solution containing the precursor particles, osmium tetroxide, and a solvent is not particularly limited, but it is generally preferable to adjust the content of osmium tetroxide to 0.05 to 3.0 moles per mole of Fe ₃ O ₄ in the precursor particles.

The present production method preferably further comprises a step of washing the composite particles (composite particle washing step) after carrying out step B. The composite particle washing step makes it possible to remove unreacted osmium tetroxide.
The washing method is not particularly limited, and may be a method of dispersing the obtained composite particles in a washing solvent (e.g., water) and recovering the solid content from the dispersion. Washing may be repeated several times.

(Method of recovering electric bacteria)
A method for recovering electric bacteria according to an embodiment of the present invention includes preparing a solution containing electric bacteria, 3,3'-diaminobenzidine, and hydrogen peroxide, staining the electric bacteria (staining process), contacting the stained electric bacteria with composite particles to form a complex between the stained electric bacteria and the composite particles (complex formation process), and recovering the complex (recovery process).

The solution used in the dyeing process contains 3,3'-diaminobenzidine and hydrogen peroxide. The content of DAB in the solution is not particularly limited, but is generally preferably 0.1 to 10 mM. The content of hydrogen peroxide in the solution is generally preferably 0.1 to 200 mM. The solution may contain a buffering agent for adjusting the pH in addition to DAB and hydrogen peroxide. Examples of the buffering agent include inorganic salts and/or organic salts, and specific examples thereof include Na ₂ HPO ₄ , KH ₂ PO ₄ , and trishydroxymethylaminomethane. The pH of the solution is preferably adjusted to a range of 6.0 to 9.0.

Examples of inorganic salts include sodium chloride, magnesium chloride, potassium chloride, calcium chloride, sodium phosphate, and ammonium sulfate.
Organic salts include sodium acetate, ammonium acetate imidazole salt, and imidazole hydrochloride.

The amount of salt in the solution is preferably 10 ⁻³ M or more, and generally, for example, 20 to 500 mM. The solution may not necessarily contain a salt.

The staining method is not particularly limited, and for example, the above solution may be added to a medium containing electrobacteria, stirred, and then held for 0.1 minutes to 1 hour. The temperature during holding is not particularly limited, but generally 5 to 50°C is preferred.
This process stains the electrobacteria and colours them red.
This colored substance is a polymer of DAB obtained by oxidative polymerization of DAB by active oxygen species generated from hydrogen peroxide by the action of electron transfer enzymes possessed by electrobacteria. In electrobacteria, a polymer of DAB is formed to cover the cell membrane.

There is no particular limitation on the method of contacting the dyed electric bacteria with the composite particles to form a complex between the dyed electric bacteria and the composite particles, but an example is a method of contacting the electric bacteria with the composite particles in a solution. In this case, there is no particular limitation on the solution used, and a solution containing water and the buffering agent already described can be used.

An example of a method for contacting the electric bacteria with the composite particles is a method of stirring a solution containing the electric bacteria and the composite particles.
During the dyeing process, hydrogen peroxide is reduced from the electrobacteria to generate reactive oxygen species, which causes DAB to undergo oxidative polymerization. The osmium oxide fixed to the composite particles specifically binds to the -CH=CH- bond in the polymer, forming a complex.

There are no particular limitations on the method for recovering the complex, and any known method for recovering magnetic particles can be used without particular limitations.

The present invention will be described in more detail below with reference to examples. The materials, amounts used, ratios, processing contents, processing procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the examples shown below.

[Synthesis of Composite Particles]
Fe ₃ O ₄ nanoparticles (hereinafter also referred to as "NP") were used as a commercial product (Aldrich, 1317-61-9, particle size 50-100 nm (SEM)). 0.06 g of NP was dispersed in 30 mL of deionized water to prepare a Fe ₃ O ₄ colloidal solution. Next, folic acid (hereinafter also referred to as "FA") was dissolved in a 5N aqueous solution of sodium hydroxide to prepare a 100 mM folic acid solution. 1 mL of this folic acid solution was added to the dispersion and stirred for 18 hours to obtain "Fe ₃ O ₄ @FA NP" in which Fe ₃ O ₄ and folic acid were combined.

Next, this "Fe ₃ O ₄ @FA NP" was washed with deionized water three times or more, and then dispersed in 30 mL of deionized water for further reaction. 0.5 mL of 1% OsO ₄ solution was added to this "Fe ₃ O ₄ @FA NP" colloidal solution with vigorous stirring for 4 hours to obtain "Fe ₃ O 4 @FA-OsO ₄ NP" in which Fe 3 O ₄ nanoparticles and osmium tetroxide were bound via folic acid. Then, "Fe ₃ O ₄ @FA-OsO ₄ NP" was washed with deionized water three times or more, and dispersed in 1 mL _{of deionized water. The final concentration of "Fe 3 O 4 @ FA} -OsO ₄ NP" in this dispersion was determined to be 34 mg/mL.

[Recovery test of S. oneidensis MR-1]
The bacterial solution was prepared by a conventional culture method. Shewanella oneidensis MR-1 was selected as an electrobacterium, and Escherichia coli was selected as a non-electrobacterium. The optical density of the bacterial solution was fixed at 1 and measured by UV-Vis (ultraviolet-visible spectrophotometry). 0.0119 g of DAB powder was dispersed in 0.22 mL of 1 M HCl (aq) by ultrasonication in the dark for 45 minutes to prepare a DAB solution.
Next, the DAB solution was diluted with 7.78 mL of Tris (pH 8) buffer (50 mM) and sonicated for 15 minutes. After that, excess DAB powder was removed with a 0.22 μm filter. DAB staining of S. oneidensis MR-1 was performed by adding the filtered DAB solution to the bacterial solution and reacting for 5 minutes. S. oneidensis MR-1 to which the DAB solution was added showed a noticeable brown color after 5 minutes of reaction. On the other hand, no color change was observed in E. coli to which the DAB solution was added. After DAB staining, the cells were washed three or more times with DM-L buffer (Defined media with 10 mM sodium lactate added).

2 μL of the cell solution with a known concentration was mixed with "Fe ₃ O ₄ @FA-OsO ₄ NP" (34 mg/mL) under vortexing for 60 minutes. Then, the cells attached to "Fe ₃ O ₄ @FA-OsO ₄ NP" were collected by magnetic attraction. The supernatant was removed, and fresh lactate-containing DM (defined media) buffer was added. This process was repeated three times to completely remove nonspecifically captured cells. These captured cells were then further analyzed by SEM imaging, colony formation, and protein quantification.
For protein quantification, the collected cells were vigorously stirred with 30 μL of lysis buffer for 10 min. The mixture was then centrifuged at 15,000 rpm for 10 min to separate the mixture pellet (magnetic nanobeads and cell debris) and protein supernatant. The protein amount in the supernatant was determined using a protein assay kit and standard measurement method.
The above results demonstrated that the electrobacterium (S. oneidensis MR-1) was specifically recovered by using "Fe ₃ O ₄ @FA-OsO ₄ NP".

Claims (10)

a magnetic nanoparticle; an organic compound bound to a surface of the magnetic nanoparticle; and osmium tetroxide bound to the organic compound ;
The composite particle for electrobacterial recovery , wherein the organic compound comprises cysteine, cystine, or folic acid . The composite particle for recovering electric bacteria according to claim 1, wherein the organic compound has a hydroxy group and an amino group, is bonded to the magnetic nanoparticles via the hydroxy group, and is bonded to the osmium tetroxide via the amino group. The composite particle for recovering electrobacteria according to claim 1 or 2, wherein the organic compound includes folic acid. The composite particles for electrobacterial recovery according to any one of claims 1 to 3, wherein the magnetic nanoparticles comprise Fe ₃ O ₄ nanoparticles. Reacting _Fe3O4 nanoparticles with _folic acid in a solvent to obtain precursor particles having folic acid bound to the surface of _{the Fe3O4} nanoparticles;
reacting the precursor particles with osmium tetroxide in a solvent to obtain composite particles in which the osmium tetroxide is bound to the folic acid. preparing a solution containing electrobacteria, 3,3'-diaminobenzidine, and hydrogen peroxide to stain the electrobacteria;
Contacting the dyed electrobacteria with a composite particle for recovering electrobacteria according to any one of claims 1 to 4 to form a composite of the dyed electrobacteria and the composite particle;
and recovering the complex. The method for recovering electrobacteria according to claim 6, further comprising washing the stained electrobacteria before contacting the stained electrobacteria with the composite particles. The method for recovering electrobacteria according to claim 6 or 7, wherein the recovery of the complex is carried out using a magnet. A composite particle in which _Fe3O4 nanoparticles and _osmium tetroxide are bound to each other via folic acid. The composite particle according to claim 9, wherein the bond is a coordinate bond.