JP2001242080A - Photophoresis method based on absorption spectrum - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new photophoresis method for separating particulates carrying no electric charge and particulates carrying the same surface charge by means of a simple operation. SOLUTION: In this photophoresis method, particulates with different absorbance in liquid are irradiated with light, and according to light absorption, a particulate with a different migration speed is detected, separated, selected or measured. A device for this method and a particulate photophoresis migration speed increasing agent, which is made of a pigment with absorbance at a wavelength of the light radiated in this method and radiates the light to the particulates with different absorbance in the liquid for increasing its migration speed based on light absorption, are available.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for separating fine particles in a liquid, and provides a new method using light energy. More specifically, the present invention is a method of irradiating light to fine particles having different absorbances in a liquid, and detecting, separating, sorting or measuring fine particles having different migration speeds due to light absorption, an apparatus therefor, and light. The present invention relates to a photophoretic velocity enhancer for increasing the degree of migration.

[0002]

BACKGROUND OF THE INVENTION Separation and characterization of particulates in liquids is one of the important themes of modern analytical chemistry. Dispersions of fine particles are ubiquitous in living systems, the hydrosphere, industrial products and their manufacturing processes, and are the subject of research in fields such as biology, biotechnology, medicine, ecology, environmental science, and industrial chemistry. Because there is. Hitherto, various electrophoretic analysis methods have been applied to fine particles in a liquid such as latex, emulsion droplets, micelles, vesicles, and semiconductors, and separation and characterization by electrophoresis have been performed. Electrophoresis, sedimentation, dielectrophoresis, and electrophoresis have been developed as such electrophoresis.

[0003] Of these electrophoretic analysis methods, the electrophoretic method and the sedimentation method have been widely applied because their electrophoretic mechanisms have already been elucidated. However, the electrophoresis method mainly uses charged fine particles, the selection of the medium is often empirical, and preliminary experiments are often required. These are not all-purpose electrophoresis methods because they have disadvantages such as the need to have them. In addition to the above-mentioned electrophoresis analysis method, by developing a new electrophoresis field and an analysis method using electrophoretic driving force, a simple separation method for difficult-to-separate fine particles and a new Information can be obtained.

In recent years, it has been reported that three kinds of polystyrene latex having different particle diameters in a fluid were separated in size by using a laser radiation pressure (Imasaka, et al.,
Anal. Chem. 1995, 67, 1763-1765.). In the early 20th century, a phenomenon in which light exerted a force on an object was discovered, but because the object was moved by the temperature gradient caused by light rather than the light itself, until light was used to move the object Spent half a century. With the advent of lasers and improvements in their performance, A. Ashkin. Discovered and reported in 1970 the phenomenon in which polystyrene latex in water moved by light irradiation by laser irradiation in the field of physics. Ashkin, A. Phys. Rev. Lett. 1970, 24, 156-1
59.). After that, T
It has become known that when a laser beam of EM ₀₀ mode is irradiated, two different phenomena are observed depending on the irradiation conditions. One is observed when the laser is strongly focused by a lens with a short focal length, and the fine particles are trapped in the focus by the gradient force of the radiation pressure of light (Ashkin, A., et a).
l., S. Opt. Lett. 1986, 11, 288-290 .; Ashkin, A, et.
al., Science 1987, 235, 1517-1520 .; Misawa, H., e.
t al., Chem. Lett. 1990, 8, 1479-1482 .; Ashkin, A.,
Biophys. J. 1992, 61, 569-582.).

[0005] This phenomenon is known as optical tweezers,
It has been put to practical use in the fields of biology and medicine as a technique for handling cells and bacteria in a non-contact and non-destructive manner. The other is a phenomenon that occurs when the laser is weakly focused or not focused at all by a lens having a long focal length. The fine particles move in the light irradiation direction due to the scattering force of the radiation pressure of the light. Although this phenomenon has great potential as a method for electrophoretic analysis of fine particles in a liquid, it has hardly been taken notice so far. The present inventors have previously observed how the refractive index and particle size of droplets of various organic droplets in water change the migration speed by laser light irradiation. (A. Hirai, H. Monjushiro, H. Watarai, Langmuir, vol.
12, 5570-5575, 1996). Such photophoresis is a phenomenon in which particles move due to scattering of irradiated light due to the difference between the refractive index of particles and that of a solution, and fine particles can be separated according to their refractive index and particle size. .

[0006] Such a method of separating fine particles by photophoresis can separate even non-charged fine particles, and has been widely used as a conventional technology for separating biological substances such as DNA and environmental substances. Unlike the electrophoresis method such as the capillary electrophoresis method, there is a great feature that it does not require skill for adjustment and measurement. However, the conventional photophoresis method separates according to the refractive index and particle size of the fine particles, and cannot be separated due to other factors, and has a drawback that the migration speed is slow. Therefore, development of a new technique of the photophoresis method is expected.

[0007]

SUMMARY OF THE INVENTION The present invention provides a novel photophoresis method. The present invention provides a novel photophoresis method capable of separating uncharged fine particles or fine particles having the same surface charge by a simple operation.

[0008]

Means for Solving the Problems The present inventors have conducted various studies on the separation and characterization of fine particles in a liquid in a photophoresis method. I found it to wake up.

The present invention relates to a method for irradiating light to fine particles having different absorbances in a liquid to detect, separate, sort or measure fine particles having different migration speeds due to light absorption. Further, the present invention can detect, separate, sort or measure fine particles that have been subjected to photophoresis by irradiation with light, a transparent container capable of storing a subject made of a liquid material containing fine particles having different absorbances, and fine particles that have been subjected to light irradiation. The present invention relates to an apparatus for irradiating light to fine particles having different absorbances in a liquid and detecting, separating, sorting, or measuring fine particles having different migration speeds due to light absorption. Furthermore, the present invention relates to an agent for enhancing the photophoresis speed of fine particles by absorbing light by irradiating light to fine particles having different absorbances in a liquid, which are composed of a dye having absorbance at the wavelength of light to be irradiated in the above method.

The present inventors have previously determined that, for various organic droplets in water, the refractive index and particle size of the droplets are proportional to the refractive index and particle size of the particles by laser light irradiation. (A.Hirai, H.Monjushiro, H.Watarai, La
ngmuir, vol 12, 5570-5575, 1996). In this study, the present inventors examined the effect of light absorption of fine particles on laser light by irradiating laser light to droplet particles in water having absorption at the laser wavelength. It has been found that the migration speed is dramatically increased as compared to fine particles having no light absorption.

The present inventors first investigated by measuring the absorption spectrum that 2-benzylpyridine to which molybdenum blue was added had absorption at a wavelength of 1064 nm of the laser beam to be irradiated. The results are shown in FIG. FIG. 1 shows a case where molybdenum blue is not added (solvent), a case where molybdenum blue is added at a concentration of 2.4 × 10 ⁻² mol / dm ³ , and a case where molybdenum blue is 7.2 × 10 ⁻² mol / dm ^3.
shows the absorbance (1mm cell) at each wavelength (nm) when added at a concentration of dm ^3. The absorbance in FIG. 1 is shown as a ratio when the absorbance of benzene is 0.00. Looking at the absorbance A at 1064 nm, which is the wavelength of the laser light used in the experiment, from FIG. 1, the case where molybdenum blue was not added (2-benzylpyridine) was used.
Has an absorbance A of almost 0, whereas the addition of molybdenum blue at a concentration of 2.4 × 10 ⁻² mol / dm ³ has an absorbance A of 0.25 (25%). The absorbance A when blue was added at a concentration of 7.2 × 10 ⁻² mol / dm ³ was found to be 0.71 (71%). The following experiment was performed using these three types of samples.

These samples were dispersed in water while applying ultrasonic waves to prepare an aqueous solution in which droplets having a particle size of 0.5 to 3 μm were dispersed. The solution containing these droplets was placed in a glass microcell, and the photophoresis of the fine particles in these solutions was observed using the apparatus shown in FIG. FIG. 2 shows an example of an apparatus for detecting, separating, sorting or measuring according to the present invention. The prepared sample is placed in the microcell 2 and irradiated with laser light from the laser light source 1, collected by the lens 3 and irradiated on the microcell 2. The behavior of the fine particles in the microcell 2 is measured by the CCD 4 through the microscope 4.
The camera 5 captures an image and the captured image is converted to a video system 6.
Was displayed.

In this experiment, C was used as the laser source.
W Nd: YAG laser (ADLAS, DIODE LASER PUMPED
Nd: YAG LASER, DPY 321) uses a wavelength of 1064 nm, and green light of 532 nm as the second harmonic is 1064 n.
No cold mirror was used because the intensity was less than 5% of the m-light. Outgoing laser beam diameter is 1.0m
m, and when irradiated as it is, light enters the entire cell containing the sample. In order to prevent this, the beam diameter was reduced using the condenser lens 3, and the diameter at the sample position was adjusted to 70 μm. The laser power was 310 mW.
The condenser lens 3 has a focal length of 6.00 cm, and a microscope 4 (Nikon, Labophot YF Type 10) is used at the focal point.
Each part was arranged so that the objective lens of 4) was in focus. The sample has an internal dimension of 100 μm × 100 μm × 50
mm microcell 2 (Vitro Dynamics Inc., Standar
d Glass Microcells) and put under the microscope xy
Placed on stage. Observation of electrophoretic behavior is performed by CCD camera 5.
(Using Sony, DXC-108) under a microscope connected, and the obtained images were recorded on a video system 6 and displayed on a monitor.

FIG. 3 shows the results of the photophoretic velocity at various particle sizes (μm) of each sample. The horizontal axis in FIG. 3 is the particle size (μm)
And the vertical axis indicates the photophoretic velocity (μm / sec). Triangles in FIG. 3 indicate the case where molybdenum blue was not added (A = 0), and diamonds indicate molybdenum blue at 2.4 × 10 4.
^-2 mol / dm ³ (A = 0.2
5), and the circles indicate molybdenum blue at 7.2 × 10
^-2 mol / dm ³ (A = 0.7
1) is shown. As is clear from these results, it can be seen that when the absorbance A of the fine particles increases, the migration speed dramatically increases. In particular, it is understood that the difference becomes remarkable when the particle size is 2 μm or more.

FIG. 4 shows the relationship between the absorbance and the photophoretic velocity when the particle size is 2 μm. The horizontal axis in FIG.
m shows the absorbance of the fine particle component in a 1 mm cell at m,
The vertical axis indicates the photophoretic speed (μm / sec). When the absorbance is about 0, the photophoretic velocity is about 15 μm / sec. When the absorbance is 0.25, the photophoretic velocity increases to about 25 μm / sec. When the absorbance is 0.71, Increase to 65 μm / sec. This result clearly shows that even when the particle diameter is the same and the refractive index is almost the same, the photophoretic speed is greatly different due to the difference in absorbance. The difference in the photophoretic speeds enables the detection of fine particles having different absorbances in accordance with the absorbance of each fine particle by light irradiation. It has been shown that fine particles having a specific absorbance can be separated or sorted, and the number of fine particles having a specific absorbance can be measured from a fine particle mixture by using the difference in photophoretic velocity.

As described above, the present invention provides a method and a device for detecting, separating, selecting or measuring fine particles by irradiating light to fine particles having different absorbances, based on the absorbance, preferably the absorbance and the particle size. To provide. Further, since the present invention can use a light absorbing agent such as a dye to increase the photophoresis speed in the photophoresis method as described above, the absorbance at the wavelength of the light to be irradiated in the photophoresis method can be increased. A light absorber having, for example, a dye such as molybdenum blue, used to irradiate light to fine particles having different absorbances in a liquid to increase the photophoretic speed of the fine particles due to light absorption, or a photophoretic speed enhancer Is provided.

BEST MODE FOR CARRYING OUT THE INVENTION

The fine particles to be detected, separated, sorted or measured in the method of the present invention may have their own specific absorbance. In the above, a light absorber that absorbs the light may be added to change the absorbance of the fine particles. The light absorber used in the method of the present invention is not particularly limited as long as it absorbs the light at the wavelength of the irradiated light and does not adversely affect the fine particle component. The degree of light absorption does not need to be the maximum absorption of the light absorbing agent used, and can change the absorbance of the fine particles when the light absorbing agent is added to the fine particles as illustrated in FIG. Should be fine. The light absorber of the present invention may be a dye, for example, molybdenum blue when the light to be irradiated is in or near the visible region, but is not necessarily colored. In addition, in order to give a difference in absorbance to the fine particles, a difference in absorbance can be given by using a different light absorber, or a difference in absorbance can be given by the concentration difference using the same light absorber. .

The method of the present invention relates to the behavior of fine particles in a liquid. The "liquid" is not particularly limited as long as it can be used as a solvent and does not absorb significantly at the wavelength of light to be irradiated. , But water is usually preferred. The water may be pure water, or may be an aqueous solution such as physiological saline. The wavelength of the light to be irradiated in the method of the present invention may be any wavelength capable of migrating the fine particles, but is preferably selected from among the absorption bands of the fine particles to be detected, separated, sorted or measured, and more preferably. The difference in the absorbance of the fine particles is a clear wavelength. If there is no significant difference in the absorbance of the fine particles themselves, it is preferable to select from among absorption bands of a light absorber, for example, a dye. Various light sources can be used, but a single wavelength laser beam is preferred.

The method of the present invention detects, separates, sorts, or measures the fine particles based on the difference in the absorbance of the fine particles. It can be seen from the results shown in FIG. 3 that the fine particles having the particle diameter and absorption of the above can also be detected, separated, sorted or measured. Therefore, the present invention provides a method for irradiating light to fine particles having different absorbances existing in a liquid to detect, separate, sort or measure fine particles having different migration speeds depending on the light absorption and the particle size of the fine particles. It is also a thing.

The present invention further provides an apparatus for implementing the above-described method of the present invention. The device of the present invention is basically a light irradiation device, a transparent container capable of storing a subject made of a liquid material containing fine particles having different absorbances, and detecting, separating, and sorting the fine particles that have been subjected to photophoresis by light irradiation. Or consists of a device that can be measured, if necessary, detection,
Special devices for separation, sorting or counting can also be provided. For example, a CCD camera can be provided on a device that can detect, separate, sort or measure electrophoresis to record migration. Further, a processing device for performing image processing on an image obtained by the CCD camera and a monitor device for monitoring the processing device can be provided.

[0021]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the present invention.

Example 1 Preparation of molybdenum blue-containing 2-benzylpyridine 12-molybdophosphoric acid (H ₃ PMo ₁₂ O ₁₄ .xH ₂
O) to an aqueous solution of O) at pH 1 by adding ascorbic acid
An aqueous solution of molybdenum blue was obtained. The obtained aqueous molybdenum blue solution was treated with 2-benzylpyridine (d = 1.06).
7, n = 1.58), washed and dried to prepare a molybdenum blue-containing 2-benzylpyridine solution.
The absorbances of the obtained molybdenum blue-containing 2-benzylpyridine solution and 2-benzylpyridine were measured by photoacoustic spectroscopy (PAS). The cell used was 1 mm. The result is shown in FIG. At a wavelength of 1064 nm, absorption spectra of about 71% and about 25% were observed.

Example 2 Preparation of Fine Particles in Water While applying ultrasonic waves to a weighing bottle containing 2.0 mL of ultrapure water, 2.0 μL of 2-benzylpyridine or a molybdenum blue-containing 2-benzylpyridine solution was micropipetted. (Eppendorf, 0.5 μL to 10.0 μL) and add o /
A w (Oil in Water) emulsion was prepared. The particle diameter of the obtained droplet fine particles was approximately 0.5 μm to 3 μm in radius. The obtained emulsion was able to form a stable emulsion for several tens minutes to several hours without adding a surfactant.

Example 3 Measurement of photophoresis Photophoresis of the emulsion obtained in Example 2 was observed using the apparatus shown in FIG. Each sample has an inner diameter of 100 × 10
0 μm, 250 × 250 μm outer diameter rectangular optical cell (Vitr
o In a microcell consisting of Dynamics Inc., Standard Glass Microcells), wavelength 1064 nm, output 310
mw, 70 μm irradiation spot diameter laser light source (cw
Nd: YAG laser (CVI LASER Corp. or ADLAS DPY
-321)). Observation of the migration behavior was performed using a microscope (Ni) connected to a CCD camera (SONY DXC-108).
kon Labophoto) and the resulting images were recorded on video and displayed on a monitor. The upper, lower, left and right of the image on the monitor corresponded to the upper, lower, left and right when the optical cell was viewed from above. A microscope objective lens having a magnification of × 40 was used. The results are shown in FIGS. The electrophoresis speed without light absorption is about 15 μm / sec, whereas the absorbance is 28 μm / sec at 25% and 6% at 71%.
The value was 5 μm / sec.

[0025]

The present invention provides a novel electrophoresis method called photophoresis based on absorbance, in which non-charged particles and surface charges that have been difficult to separate and sort by conventional electrophoresis are the same. Even particles can be separated by the method of the present invention by differences in absorbance. In addition, the method and apparatus of the present invention are relatively simple and can measure the behavior of particles in real time without requiring complicated adjustment of the apparatus and skill in measurement in measurement. Further, the movement of the particles can be easily controlled by turning on / off the light source. The present invention also provides use of a light absorbing agent such as a dye which absorbs light, to increase the photophoretic speed of fine particles due to light absorption, or to provide a new use as a photophoretic speed enhancer. .

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the results obtained when molybdenum blue was not added and when molybdenum blue was added at various concentrations.
Absorbance (1) at each wavelength (nm) of benzylpyridine
mm cell).

FIG. 2 shows an example of an apparatus for detecting, separating, sorting or measuring according to the present invention.

FIG. 3 shows the results of photophoretic velocities at various particle sizes (μm) of each sample using 2-benzylpyridine. The horizontal axis in FIG. 3 shows the particle diameter (μm), and the vertical axis shows the photophoretic velocity (μm / sec). Triangles in FIG. 3 indicate the case where molybdenum blue was not added (A = 0), and diamonds indicate molybdenum blue at 2.4 × 10 ⁻² mol / d.
The case where A was added at a concentration of m ³ (A = 0.25) is shown, and the circles indicate molybdenum blue at 7.2 × 10 ⁻² mol / dm.
^The case where A was added at a concentration of ³ (A = 0.71) is shown.

FIG. 4 shows the relationship between the absorbance and the photophoretic speed when the particle size is 2 μm when molybdenum blue is added to ^2- benzylpyridine at a concentration of 7.2 × 10 ⁻² mol / dm ^3. Show. The horizontal axis in FIG. 4 shows the absorbance of the fine particle component in a 1 mm cell at 1064 nm, and the vertical axis shows the photophoretic speed (μm / sec).

[Explanation of symbols]

 Reference Signs List 1 laser light source 2 microcell 3 lens 4 microscope 5 CCD camera 6 video system

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 21/17 G01N 21/17 A

Claims (13)

[Claims] 1. A method for irradiating light to fine particles having different absorbances in a liquid, and detecting, separating, sorting or measuring fine particles having different migration speeds due to light absorption. 2. The method according to claim 1, wherein the absorbance of the fine particles is varied depending on the dye. 3. The method according to claim 2, wherein the absorbance differs depending on the concentration difference of the dye. 4. The method according to claim 1, wherein the dye is molybdenum blue.
A method according to any one of claims 1 to 3. 5. The method according to claim 1, wherein the wavelength of the light to be irradiated is selected from the absorption bands of the fine particles to be detected, separated, sorted or measured. 6. The method according to claim 5, wherein the wavelength of the irradiating light is selected from the absorption bands of dyes added to the fine particles to be detected, separated, sorted or measured. 7. The method according to claim 1, wherein the light is a laser light. 8. The method according to any one of claims 1 to 7, wherein the method according to any one of claims 1 to 7, further comprising detecting, separating, sorting or measuring according to the particle size of the fine particles. 9. A light irradiation device, a transparent container capable of storing a subject made of a liquid material containing fine particles having different absorbances, and a device capable of detecting, separating, sorting, or measuring fine particles photophoresed by light irradiation. An apparatus for irradiating light to fine particles having different absorbances in a liquid and detecting, separating, sorting or measuring fine particles having a different migration speed due to light absorption. 10. The apparatus according to claim 9, wherein the light is a laser light. 11. The device according to claim 9, wherein the device capable of detecting, separating, sorting, or measuring fine particles electrophoresed by light irradiation is a device having a CCD camera. 12. The apparatus according to claim 11, further comprising a processing device for image-processing an image obtained by the CCD camera. 13. A fine particle by absorbing light by irradiating light to fine particles having a different absorbance in a liquid, comprising a dye having an absorbance at the wavelength of the light to be irradiated in the method according to any one of claims 1 to 8. Photophoresis speed enhancer.