JP2558908B2 - Method for producing magnetic fine particles - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing magnetite magnetic fine particles on which an organic coating layer for labeling a biomaterial is formed.

[Prior Art and its Problems] Since magnetic fine particles can be selectively separated, collected and moved by a magnetic field after being bound to a specific object, they have been conventionally used as a magnetic fluid or a magnetic ink. Recently, use as a carrier for biopolymers, cells, viruses, or drugs has been proposed.

Magnetic fine particles for the purpose of binding to minute objects such as cells and viruses are desired to have improved characteristics as compared with those conventionally used for magnetic fluids.

For example, in the case of magnetic particles considering binding to a virus, it is desirable that the particle size is controlled to about 100Å and the particle size distribution is close to monodisperse. This particle size is a value when considering magnetite (Fe ₃ O ₄ ), which is equivalent to the size of a virus (it easily binds to a virus) and also satisfies the magnetic characteristics. Further, it is necessary that an appropriate organic coating layer is formed on the surface of the individual magnetic fine particles so that the magnetic fine particles do not easily aggregate, and that they are uniformly dispersed in the solution.

As a method for producing the above-mentioned magnetic fine particles for capturing a micro biomaterial, attempts have been made to synthesize iron oxide in an aqueous solution of dextran, which is a polysaccharide, or liposome, which is a lipid.

The present inventor also previously mentioned in the patent application that by adding ammonia water to an aqueous dextran solution in which the molar ratio of divalent and trivalent iron ions is 2Fe ³⁺ <Fe ²⁺ , an average particle size of 10
It has been reported that Fe ₃ O ₄ fine particles of 0 Å or more can be synthesized. The magnetic fine particles thus obtained are
Since its surface is covered with dextran, the remanent magnetization is zero and it has been clarified that it exhibits superparamagnetism.

The above-mentioned production method was satisfactory in controlling the magnetic properties and average particle diameter of the obtained magnetic fine particles, but it is not always satisfactory in particle size distribution of the magnetic fine particles and uniform dispersibility in the solution. Was not.

Conventionally, magnetic particles for labeling biomaterials are RS Molday and Ma
It was produced by the method described in ckenzie, J. Immunological Methods 52, p353, 1982.

This method involves adding ammonia water to an aqueous solution in which dextran, FeCl ₃ and FeCl ₂ are dissolved to form an aqueous solution.
The pH is increased and the magnetite fine particles are generated in the solution. However, this method has a disadvantage that the pH of the portion where the ammonia water is dropped is changed abruptly, and only a fine particle dispersion liquid having poor uniformity is obtained.

On the other hand, in order to realize a uniform reaction, in magnetite synthesis for a recording medium, a method of adding urea to an aqueous solution of iron chloride in advance and raising the temperature to decompose it to generate ammonia has been attempted. However, when this method using urea is applied to the synthesis method of the above-mentioned document,
There was a problem that 300 to 400 Å magnetite particles were precipitated at once, and dextran-coated particles could not be obtained. This is because the decomposition of urea requires a temperature of 80 to 90 ° C., which is close to the boiling point of water, so that the particle size of the generated fine particles cannot be kept small. The magnetite thus obtained no longer exhibits superparamagnetism, so that it cannot be used as magnetic fine particles for labeling.

An object of the present invention is to provide magnetic microparticles having a uniform particle size and particle size distribution, which are suitable for use as a microbiomaterial label that can be uniformly dispersed in a solution.

[Means for Solving the Problems] In the production method of the present invention, divalent iron ions and / or 3
A method for producing magnetic fine particles having an organic coating layer formed on the surface by increasing the pH of a raw material aqueous solution containing valent iron ions and an organic coating layer forming material, the total iron ion concentration of the raw material aqueous solution Of 6 × 10 ^-2 mol / or less and urea is added to the raw material aqueous solution, and the urea is pyrolyzed to increase the pH of the raw material aqueous solution to produce magnetite magnetic fine particles with a particle size of 150 Å or less. It was taken as a solution.

[Operation] The production method of the present invention is to uniformly increase the pH of the raw material aqueous solution by ammonia generated by thermal decomposition of urea to synthesize magnetite fine particles.

Further, by limiting the concentration ratio of the sum of the divalent and trivalent iron ion concentrations in the raw material aqueous solution and the organic coating layer forming material, it is possible to prevent the particle size of the obtained magnetic fine particles from excessively increasing and precipitating.

Examples of the organic coating layer forming material used in the production method of the present invention include polysaccharides such as dextran, lipids such as liposomes, and aqueous solution polymers such as polyvinyl vinyl alcohol.

The solubility of the organic coating layer forming material in water is not generally large, and for example, in the case of dextran, it is about 25 g dextran / 100 g H ₂ O. Therefore, in the present invention, the concentration of the organic coating layer forming material is set to a constant value close to the solubility limit at which it can be dissolved in water, and a practical method of setting the concentration of iron ions in the optimum concentration range is adopted.

Further, in the present invention, iron ions contained in the raw material aqueous solution are
It is not particularly limited as long as it can precipitate the magnetite fine particles by increasing the pH, and may contain at least one of divalent iron ion (Fe ²⁺ ) and trivalent iron ion (Fe ³⁺ ). Good.

[Examples] Hereinafter, the present invention will be described in detail with reference to Examples.

(Example 1) The horizontal axis represents the heating time of the raw material aqueous solution, and the vertical axis represents the pH change of the aqueous solution. The results are shown in FIG.

 The manufacturing conditions here are as follows.

Iron ion concentration: 4.4 × 10 ^-3 mol / Fe ³⁺ : Fe ²⁺ = 1: 2 Urea concentration: 10 g / Dextran concentration: 10 g / Put this raw material aqueous solution in a sealed Teflon container and
It was placed in an oil bath at 110 ° C, taken out after a certain period of time, and analyzed one after another.

Here, the dextran concentration is set low in order to show the generation, growth, and sedimentation of magnetite particles produced by the conventional method. The manufacturing conditions in the above-mentioned Molday document are iron ion concentration: 2.2 × 10 ⁻¹ mol / dextran concentration: 125 g /.

Even if urea is added to an aqueous solution of this concentration and the same experiment is performed, the same phenomenon as the result shown in FIG. 1 is observed.

About 50 minutes after heating, the yellow color of the iron chloride aqueous solution changes to black. At this time, the solution was taken out, dried and analyzed by X-ray diffraction, and it was found that magnetite having a particle size of about 70Å was produced. Further heating,
Fine particles settle down after 1 hour.
These fine particles continue to grow even after settling, and the particle size increases to 175Å after 1 and a half hours and to 228Å after 2 hours. Under this heating condition, the pH value becomes almost saturated after about 2 hours.

In order to prevent precipitation at the iron ion concentration of Example 1 (4.4 × 10 ⁻³ mol /), at least a dextran concentration of 50 was used.
It turned out to be g /.

Further, the urea concentration was set to 10 g / in Example 1, which is because the concentration of iron chloride is relatively low. When the magnetic fine particles are produced by the production method of the present invention, since the aqueous iron chloride solution is acidic, it is necessary to appropriately add urea according to the iron ion concentration.

The results are shown in FIG. 2, in which the horizontal axis represents the molar ratio of urea and iron ions and the vertical axis represents the pH of the iron chloride aqueous solution after heating. It can be seen that when the molar ratio of urea to iron ion exceeds 50, the pH becomes 8 or more, and then the pH becomes saturated. Therefore,
The molar ratio of urea and iron ions in the raw material aqueous solution is at least
It turns out that 50 or more are necessary. The molar ratio of urea to iron ions in Example 1 was 189, which satisfies the condition shown in FIG.

Example 2 An aqueous solution sample in which dextran-coated magnetite fine particles were suspended was prepared by the manufacturing method of the present invention and a conventional manufacturing method (method described in Molday's literature). Then, it was measured how uniformly the magnetite fine particles were dispersed in each aqueous solution sample without agglomeration. Hereinafter, the details will be described in the order of manufacture and measurement.

First, an aqueous solution sample was prepared by the manufacturing method of the present invention.

Iron ion concentration (Fe ³⁺ : Fe ²⁺ = 1: 2): 2.2 × 10 ^-2 mol /,
1.1 × 10 ^-2 mol /, 7.3 × 10 ^-3 mol /, 4.4 × 10 ^-3 mol /
, 2.2 × 10 ⁻³ mol / aqueous solution, and 100 g / dextran (weight average molecular weight: 40,000) and 50 g / urea were respectively added to prepare a raw material aqueous solution. Each of the raw material aqueous solutions was placed in a Teflon container capable of being sealed and immersed in an oil bath at 110 ° C. for 2 hours. Then, each raw material aqueous solution was dialyzed and diluted with pure water so that the iron ion concentration was 2.2 × 10 ⁻³ mol / all.

 Next, an aqueous solution sample was prepared by the conventional method.

Iron ion concentration (Fe ²⁺ : Fe ³⁺ = 1: 2): 4.4 × 10 ^-2 mol / 100 ml of aqueous solution, 25 g of dextran (weight average molecular weight: 4
0000) was added to make a raw material aqueous solution. Add to this raw material aqueous solution 7.
100 ml of 5% aqueous ammonia was added dropwise. At the time of dropping, sufficient stirring was performed while maintaining the solution temperature at 5 ° C. afterwards,
Aged at 60 ° C for 1 hour. This solution was dialyzed and diluted with pure water so that the iron ion concentration would be 2.2 × 10 ⁻³ mol / for comparison with the sample produced by the production method of the present invention at the time of measurement.

The aqueous solution sample obtained by the present invention was compared with the aqueous solution sample obtained by the conventional method.

It is not always clear how to measure the dispersibility of the organic substance-coated ultrafine particles. A commonly known method is transmission electron microscopy (TEM)
Although the state of aggregation of particles is observed by using the above method, it is impossible to directly measure the aqueous solution. Therefore, a method of scooping particles in an aqueous solution onto a mesh once and estimating the state in the aqueous solution from the aggregated state of the particles on the mesh is adopted. Since this method is indirect and difficult to express as a numerical value, here, by correlating the angle dependence of light scattering shown below, the result of dynamic light scattering, and the observation result of TEM, It was made possible to more directly grasp the dispersion state of fine particles.

FIG. 3 shows a schematic diagram of the light scattering measurement system. This measurement method uses a wavelength of 0.44 in a cell filled with an aqueous solution containing fine particles.
The amount of scattered light is measured by irradiating with 8 μm light (argon laser) and changing the angle. This scattered light has the same frequency as the incident light, and such a phenomenon is called static light scattering. Then, the static light scattering of a benzene solution or the like is measured in advance as a standard, and the amount of scattering of the sample is determined by comparing with that value.

Specifically, R (θ) = (r ² Iθ) / (VI ₀ ) r: distance from scattering center to observation surface, Iθ: scattered light intensity, I ₀ : incident light intensity, θ: scattering angle, V : Calculated as the Rayleigh ratio represented by the scattering volume.

FIG. 4 shows the Rayleigh ratio with respect to the scattering angle of each aqueous solution sample. The numerical values in the figure indicate the iron ion concentration of the raw material aqueous solution of each aqueous solution sample, and the concentration of each aqueous solution sample is unified to 2.2 × 10 ^-3 mol / when converted to iron ion. From FIG. 4, it can be seen that as the iron ion concentration of the raw material aqueous solution decreases, the amount of scattering also decreases and the forward scattering also decreases. All the aqueous solution samples are not shown because the figure becomes complicated, but the iron ion concentration of the raw material aqueous solution is 2.2 × 10 ^-2 mol / (1/1 of the conventional example).
In the sample aqueous solution (concentration of 0), sedimentation was observed in a part of the particles, and the scattering was almost equal to or slightly higher than the result of the conventional method. The iron ion concentration in the raw material aqueous solution is 1.1 × 10 ^-2.
From (concentration of 1/20 of the conventional example) to 2.2 × 10 ^-3 mol / (concentration of 1/100 of the conventional method), a constant proportional relationship was found between the iron ion concentration and the amount of scattering.

Fine particles in these aqueous solution samples were placed on the mesh for a certain period of time, and then water was absorbed with a filter paper and observed with TEM (magnification of about 20,000 times). It became clear that there was a correlation between them. From this, it was clarified that the dispersibility of the organic substance-coated fine particles in the solution can be evaluated by static light scattering.

Further, FIG. 4 also shows, for comparison, the scattering characteristics of a gold fine particle suspension aqueous solution (commercially available product), which is well known for its monodispersion. The Rayleigh ratio of this gold fine particle suspension aqueous solution is 2.2 × 10 ⁻³ mol / mL of the aqueous solution sample according to this example.
The feature is that it is the closest to the example of (1) and there is almost no forward scattering.

From this, it was clarified that by applying the production method of the present invention, magnetite fine particles in the same dispersed state as gold fine particles can be obtained.

Further, in order to measure the particle size distribution of fine particles more directly, a dynamic light scattering method is known. In this dynamic light scattering method, light with a frequency different from that of the incident light is observed due to the Doppler shift in the light scattered when the fine particles in the solution in Brownian motion are irradiated with laser light. The wavelength of incident light is approximately 0 /
A particle size of 100 (nm order) can also be evaluated.

Next, the sample aqueous solution was subjected to ultrafiltration to sufficiently remove excess dextran, and then the particle size distribution of dextran-coated magnetite fine particles was measured by a dynamic scattering method.
The measurement result is shown in FIG.

From FIG. 5, it was found that as the initial concentration of iron ions in the raw material aqueous solution increased, the particle size of the obtained fine particles also increased and the distribution thereof became wider. It is considered that this does not show the particle size of one fine particle, but shows the apparent particle size when some fine particles aggregate. This result supports the above-mentioned tendency of static light scattering and the TEM observation result.

Example 3 An experiment was conducted under the following conditions in order to clarify the effect of the concentration of dextran added to the raw material aqueous solution on the dispersibility of fine particles.

Iron ion concentration of raw material aqueous solution 2.2 × 10 ^-3 mol / Fe ³⁺ : Fe ²⁺ = 1: 2 Urea addition amount: 25g / Dextran concentration of 25g /, 50g / , 100 g /, and the scattering characteristics of the aqueous solution samples obtained are shown in FIG. From FIG. 6, it was confirmed that the light scattering decreased and the dispersion characteristics improved as the dextran concentration increased. It was found that by setting the dextran concentration to about 100 g / or more, a suspension of magnetite fine particles having a Rayleigh ratio of 5 × 10 ⁻⁵ cm ⁻¹ or less and excellent in dispersibility (like gold fine particles) can be obtained.

Further, FIG. 7 shows changes in the amount of light scattering when the amount of urea added to the raw material aqueous solution was changed. The conditions for producing the raw material aqueous solution are as follows.

Iron ion concentration of raw material aqueous solution 2.2 × 10 ^-3 mol / Fe ³⁺ : Fe ²⁺ = 1: 2 Dextran concentration: 100g / As shown in Fig. 7, the higher the urea concentration, the better the dispersibility and the less light scattering the aqueous solution. It turned out to be obtained.

Example 4 In order to clarify what kind of particle size magnetite was synthesized by the manufacturing method of the present invention, an experiment was conducted with the following parameters.

Dextran concentration (molecular weight 40,000): 125g / (Molda
y value in literature), 250 g / urea concentration: 50 g / iron ion concentration of raw material aqueous solution: 10 ^-3 to 10 ^-1 mol / Fe ³⁺ : Fe ²⁺ = 1: 2 The diffraction peak due to magnetite was measured by X-ray diffraction, and the method (SCherrer's formula) of determining from the half-value width was used. However, when the initial concentration of iron ions is on the order of 10 ^{-3, the} amount of magnetite produced is too small compared to dextran and it becomes difficult to measure by X-ray, so the operation of concentrating the prepared solution by ultrafiltration is performed. After that, it was dried at 40 ° C. to obtain a measurement sample. By this ultrafiltration process, dextran not bound to magnetite (called free dextran)
Are removed. FIG. 8 shows the X-ray diffraction pattern of the sample when the initial iron ion concentration was changed. The large halo pattern is derived from dextran, and the magnetite peak appears in the form of an overhang. The particle size was evaluated using the largest diffraction peak (311). The upper two diffractograms in FIG. 8 are those of the sample uniformly dispersed in the solution, and the lower two diffractograms are those of the precipitated sample. In the precipitated magnetite, it can be seen that the organic coating layer of dextran is very thin.

In addition, the horizontal axis represents the initial concentration of iron ions, and the vertical axis represents the particle size of the produced magnetite fine particles.
As shown in the figure. In Figure 9, white circles have a dextran concentration of 125 g.
In the case of /, the black circles represent the case of 250 g /. The particle size increases as the iron ion concentration of the raw material aqueous solution increases, and settling starts when the particle size becomes constant. The particle size tends to decrease as the dextran concentration increases, but as shown by the broken line in FIG. 9, the particle size at which precipitation begins is 150 Å, which does not change depending on the dextran concentration. Further, the particle size of the precipitated fine particles also increases as the iron ion concentration of the raw material aqueous solution increases.

The solubility of dextran in water is almost limited to 250 g /. Also, as mentioned above, the iron ion concentration is 4.4 × 1
A dextran concentration of at least 50 g / is necessary to prevent the magnetite fine particles from settling under the condition of 0 ^-3 mol /. In order to obtain an excellent dispersion type solution, a dextran concentration of 100 g / or more is required as shown in FIG. Therefore, in order to obtain an aqueous solution having good dispersibility without the magnetite fine particles settling, a dextran concentration of about 100 to 250 g / is optimal. It is also understood that the iron ion concentration in the raw material aqueous solution must be set to 6 × 10 ^-2 mol / or less (when the dextran concentration is 250 g /) so that the magnetite fine particles do not settle.

Example 5 Influenza virus was labeled using the dextran-coated magnetite fine particles produced by the production method of the present invention, and these were collected by applying a magnetic field and then observed by a transmission electron microscope (TEM). The specific labeling method is as follows. The dextran sugar chain was oxidized with periodic iodine (eg, NaIO ₄ ) to generate an aldehyde group, on which rabbit hyperimmune serum was purified to obtain Ig.
G antibody was coupled. Figure 10 shows the I thus obtained
It is an electron micrograph (100,000 times) of gG antibody-dextran-coated magnetite fine particles. It can be seen that the dispersed dots that appear black are individual fine particles (magnetite looks like black dots) and are dispersed very uniformly.

Then add the influenza virus sample (antigen)
Antigen-antibody reaction was carried out by incubating at 2.5 ° C for 2.5 hours. 11th
The figure shows an electron micrograph (100,000 times) after collecting magnetic particles labeled with this influenza virus by a magnetic field.
Is. It can be seen that a large number of magnetic substance-labeled antibodies (which appear black) are gathered and bound around around a dozen or so particle diameters of influenza virus having a diameter of about 100 nm.

[Advantages of the Invention] As described above, according to the method for producing magnetic fine particles of the present invention, it is possible to obtain magnetic fine particles having a particle size controlled to 100Å to 150Å and excellent dispersibility in a solution. it can.

Further, the production method of the present invention, by appropriately selecting the iron ion concentration and dextran concentration in the raw material aqueous solution,
It is possible to control the particle size of the obtained magnetic fine particles as well as the dispersibility. Therefore, when the production method of the present invention is applied to the medical field such as a labeling material in the immunoassay field and a carrier material for a drug delivery system, it is more useful by using magnetic properties and dispersion properties that cannot be achieved by conventional magnetic fine particles. Can be demonstrated.

[Brief description of drawings]

Figure 1 shows the heating time of the raw material aqueous solution and the thermal decomposition of urea.
Fig. 2 is a graph showing the change in pH, Fig. 2 is a graph showing the relationship between the molar ratio of urea and iron ions in the raw material aqueous solution and the change in pH, and Fig. 3 is for knowing the dispersion state of magnetic particles. Fig. 4 is a schematic diagram showing the light scattering measurement system of Fig. 4, Fig. 4 is a graph showing the Rayleigh ratio with respect to the scattering angle of magnetic fine particles, and Fig. 5 is the particle size distribution of dextran-coated magnetite measured by the dynamic light scattering method. The graph showing the results, FIG. 6 is a graph showing the change of the Rayleigh ratio of the magnetic fine particle dispersion solution when the dextran concentration of the raw material aqueous solution was changed, and FIG. 7 was the amount of urea added. 8 is a graph showing the change in dispersibility of the magnetic fine particles, FIG. 8 is a graph showing the X-ray diffraction pattern of the magnetic fine particles when the iron ion concentration in the raw material aqueous solution was changed, and FIG. 9 is the raw material. Iron ion concentration in aqueous solution Graph showing the relationship of the particle size of the magnetic fine particles, Fig. 10 is an electron micrograph showing the particle structure of the IgG antibody-dextra-coated magnetic particles, and Fig. 11 shows the results of binding a magnetically labeled antibody to influenza virus. It is an electron micrograph which shows the particle structure of a particle | grain.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Mitsutoshi Hoshino 1-1-6 Uchisaiwaicho, Chiyoda-ku, Tokyo Nippon Telegraph and Telephone Corporation (72) Hiroko Mizutani 6-11 Udagawa-cho, Shibuya-ku, Tokyo ( 56) References JP-A-3-137022 (JP, A) JP-A-3-141119 (JP, A) JP-A-57-175734 (JP, A)

Claims (1)

(57) [Claims] 1. A magnetic fine particle having an organic coating layer formed on its surface by increasing the pH of a raw material aqueous solution containing divalent iron ions and / or trivalent iron ions and an organic coating layer forming material. The method is to set the total iron ion concentration of the raw material aqueous solution to 6 × 10 ^-2 mol / or less, add urea to the raw material aqueous solution, and thermally decompose the urea to increase the pH of the raw material aqueous solution.
A method for producing magnetic fine particles, which comprises producing magnetite magnetic fine particles having a particle size of 150Å or less.