JP4317919B2 - How to create data for crystal characterization - Google Patents

Description

The present invention relates to an organic crystal, and in particular about the making how the sample used when evaluating the characteristics of the polymer crystals.

  In recent years, research on organic materials having high performance and high performance exceeding the characteristics of inorganic materials has attracted attention, and development of next-generation organic devices has been carried out. For this purpose, development of new organic materials is indispensable. In fields such as applied chemistry, the newly synthesized materials are crystallized, and the molecular three-dimensional structure is determined by X-ray crystal structure analysis to estimate its function. . In addition, when a crystal is made into a device, it is necessary to accurately evaluate various physical characteristics such as crystal quality and the state of the crystal surface. On the other hand, post-genome research called proteome has become popular, and what is particularly noted is research that attempts to unravel the three-dimensional structure of proteins, which is called structural genomic science. Protein structure and function analysis is an important research field in life sciences, and it is expected to produce results that are directly linked to disease treatment and drug discovery and lead to the elucidation of life phenomena. Crystals are required. Thus, in order to evaluate the characteristics of various organic materials, crystals of the materials are widely used.

  In many organic materials, crystallization conditions and growth conditions for obtaining a high-quality single crystal have not been established. Therefore, it is very difficult to control crystal precipitation and subsequent growth control. Therefore, it is difficult to grow only an ideal single crystal, and it may be polycrystallized or a crystal with defects may be obtained. Yes. Further, similarly to the growth of inorganic crystals and organic low-molecular crystals, crystals having a shape reflecting the molecular structure and growth conditions are grown, so the crystal shape may be unsuitable for measurement.

  For example, when performing X-ray crystal structure analysis, which is one of the characteristics evaluation of crystals, the crystal in the growth vessel is usually taken out, held by a crystal holder, and placed on the stage of the X-ray diffractometer, X-ray irradiation is performed. At that time, since it is difficult to maintain and maintain the crystal quality of the organic crystal, the crystal held in the crystal holder is not an ideal single crystal suitable for measurement, or the crystal is frozen in the low temperature state with the crystal holder frozen. May be measured. Specifically, there are a case where a plurality of single crystals are contained in the crystal holder, a case where a crystal in the crystal holder contains defects, a case where measurement is performed while spraying liquefied nitrogen or the like. When measurement is performed in such an environment, X-rays pass through substances other than the measurement target, and a part of the X-rays are absorbed and attenuated by these substances, and from other than the measurement target. There is a problem that reflection is included in the measurement data.

  The present invention has been made to solve such problems, and in a sample used for characteristic evaluation of an organic crystal, a substance other than a crystal that performs characteristic evaluation that causes a decrease in measurement accuracy is removed or measured. It is an object of the present invention to provide a method for producing a sample for characteristic evaluation so as not to affect the above and a method for characterizing a crystal using the sample.

A first invention for achieving the above object is a method for preparing a sample used for evaluating the characteristics of an organic crystal, which contains the organic crystal by freezing the organic crystal in a culture solution containing the organic crystal. Preparing a culture solution to be used, holding the culture solution containing the organic crystal by a holding means , and irradiating at least a part of the substance other than the evaluation target existing in the vicinity of the organic crystal to be evaluated with ultraviolet light Thus, removal, modification, or refinement is performed.

  In the present invention, among the sample including the crystal and the holding means, a substance other than the evaluation target that causes a decrease in accuracy in the property evaluation existing in the vicinity of the evaluation target is removed by irradiating with ultraviolet light, They are modified or refined. In the present specification and claims, the term “modification” refers to reducing the influence on characteristic evaluation, for example, by making a crystal amorphous. The “substance other than the evaluation target” refers to all substances including the organic crystal other than the evaluation target existing in the vicinity of the organic crystal to be evaluated, the holding means, and the attached matter. However, the surrounding gas does not belong to this. Further, “miniaturization” refers to reducing the influence on characteristic evaluation by miniaturizing a crystal. Here, “characteristic evaluation” is a concept that includes so-called well-known physical, chemical, and mechanical characteristic evaluation in general. As described above, when growing an organic crystal, it is difficult to grow an ideal single crystal, so if the crystal holder contains multiple single crystals or if the crystal in the crystal holder is defective. May be included. In addition, in the case where crystals are held by freezing in a state containing a crystal growth solution such as water such as protein or an organic salt, the frozen solution around the crystals is unnecessary for measurement. According to the present invention, since the influence of substances other than the evaluation target on the result of the crystal characteristic evaluation among the samples including the crystal and the holding means is reduced in this way, accurate characteristic evaluation is possible. It is. Moreover, the surface or the surface layer can be accurately evaluated by exposing the crystal surface.

  In the present invention, ultraviolet light is used because chemical bonds can be easily broken by using ultraviolet light having a large photon energy, so that removal and modification can be easily performed. is there. Crushing occurs when ablation occurs due to light irradiation, and the material around the part irradiated with light is mechanically broken by the impact. In this case as well, ultraviolet light having a large photon energy is used. This is because it is preferable.

A first related form for achieving the object is the first invention, wherein the characteristic evaluation of the organic crystal is characteristic evaluation of an organic crystal using a diffraction phenomenon of radiation, and the ultraviolet light is At least a part of the irradiated region is a portion where the radiation passes through the sample.

In this related form , the influence of substances other than the evaluation target existing in the part where the radiation used for characteristic evaluation passes on the measurement result is reduced, so that the absorption of radiation to be irradiated is suppressed and also due to scattering. Noise is reduced, and accurate characteristic evaluation is possible. The radiation referred to in the present specification and claims includes X-rays, electron beams, neutron beams, and the like. Examples of the characteristic evaluation using the diffraction phenomenon of radiation include crystal structure analysis and X-ray topography.

A second related form for achieving the object is the first related form , and the characteristic evaluation of the organic crystal is a crystal structure analysis.

  In the crystal structure analysis, substances other than the organic crystal to be evaluated existing in the portion through which the synchrotron radiation passes are basically all factors that cause a decrease in measurement accuracy. By applying the present invention, high accuracy is achieved. The structural analysis can be carried out. In addition, structural analysis may not be possible if a sample contains a plurality of organic crystals, but it is not necessary by irradiating ultraviolet light to unnecessary crystals other than the crystal to be evaluated. The desired structural analysis can be performed by eliminating the influence from the crystal.

A second invention for achieving the above object is the first invention, characterized in that the surface or internal state of the organic crystal is evaluated using a probe in contact with or close to the organic crystal. The surface of the organic crystal is exposed by removing the holding means and the culture solution before and after the organic crystal .

  In the characteristic evaluation of an organic crystal, when the characteristic evaluation is performed by bringing the probe into contact with or close to the evaluation target, accurate characteristic evaluation may not be performed unless the organic crystal to be evaluated is exposed. In the present invention, since a substance other than the evaluation target existing in the vicinity of the organic crystal to be evaluated is removed by irradiating with ultraviolet light, a desired evaluation can be performed. Examples of such characteristic evaluation using a probe include a scanning probe microscope including a scanning tunnel microscope and an atomic force microscope. The reason for using ultraviolet light is the same as that explained in the description of the first invention.

A third related form for achieving the object is the first invention, wherein the characteristic evaluation of the organic crystal is characteristic evaluation of the organic crystal using absorption, interference, or scattering of electromagnetic waves, At least a part of the region irradiated with the ultraviolet light is a part through which the electromagnetic wave passes through the sample.

In this related form , substances other than the evaluation target existing in the part where the electromagnetic wave used for characteristic evaluation passes are removed, modified, or miniaturized by irradiating with ultraviolet light. The influence on the measurement result can be reduced, and the characteristics of the crystal can be accurately evaluated. Examples of such characteristic evaluation using electromagnetic waves include absorption spectrum measurement and photoluminescence measurement. The reason for using ultraviolet light is the same as that explained in the description of the first invention.

A fourth related form for achieving the object is any one of the first to third related forms , wherein the ultraviolet light is ultraviolet laser light. .

  By using ultraviolet laser light, high irradiation illuminance can be obtained, and removal, modification, or miniaturization can be easily performed. In addition, since the irradiation position can be controlled with high accuracy, an effect can be given only to the target location.

A fifth related form for achieving the object is the fourth related form , characterized in that the ultraviolet laser beam is a short pulse ultraviolet laser beam having a pulse width of 100 ns or less. is there.

  When removing a substance, it is removed by breaking the chemical bond using ultraviolet light as described above. In this case, if heat is also generated, the organic crystal to be evaluated is damaged. There is a fear. If short pulse ultraviolet laser light having a pulse width of 100 ns or less is used, heat generation can be suppressed, and the substance can be removed without damaging the peripheral portion.

  When the material is pulverized, the material around the irradiated portion is mechanically broken by the impact generated by ablation. Therefore, it is desirable to use light that can give a large impact locally, and it is preferable to use ultraviolet light having a short pulse width and high energy density.

A sixth related form for achieving the object is any one of the first to fifth related forms , wherein the organic crystal is a resin, protein, saccharide, lipid, organic supramolecular complex, and Among the nucleic acids, it is at least one crystal.

The first to fifth related embodiments are particularly suitable methods for evaluating the physical properties of polymer crystals in which the growth of high-quality single crystals is particularly difficult and the crystals are easily damaged.

A seventh related form for achieving the object uses a characteristic evaluation sample manufactured by the method for producing a crystal characteristic evaluation sample of any one of the first to sixth related aspects. This is a crystal property evaluation method characterized by performing crystal property evaluation.

In this related form , it is possible to suppress the influence of substances other than the evaluation object and perform the characteristic evaluation of the organic crystal, and as a result, it is possible to obtain a more accurate evaluation result.

FIG. 1 is a diagram schematically showing an example of the first embodiment of the present invention.
FIG. 2 is a diagram schematically showing an example of the second embodiment of the present invention.
FIG. 3 is a diagram showing the results and photographs of the first embodiment of the present invention.
FIG. 4 is a diagram showing the results and photographs of the second embodiment of the present invention.

  Hereinafter, an example of an embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a diagram schematically showing an example of the first embodiment of the present invention. As shown in (a), when performing the characteristic evaluation, the organic crystal 1 is picked up by the loop-shaped holder 2 together with the culture solution 3 used for the growth, and the inside of the holder 2 is absorbed by the surface tension of the culture solution 3. Retained. In this state, the organic crystal 1 and the culture solution are frozen, and the holder 2 and the culture solution 3 before and after the organic crystal are removed by irradiation with ultraviolet light (b). In the figure, the holder 2 and the culture solution 3 above the organic crystal 1 are also removed, but this portion need not necessarily be removed.

  By doing in this way, since it does not receive the influence by the holder 2 and the culture solution 3 before and behind an organic crystal, the bad influence in characteristic evaluation reduces.

  The cutting and removal method is performed by irradiating and scanning with ultraviolet light along the line to be cut, cutting by ablation, or irradiating all the parts to be removed with ultraviolet light and removing by ablation. . At that time, as described above, it is preferable to use ultraviolet laser light as ultraviolet light, and it is preferable to use short pulse ultraviolet laser light having a pulse width of 100 ns or less. In the embodiments and examples of the present invention, the culture medium 3 and the holder 2 correspond to “holding means” in the claims and the disclosure section of the invention.

  FIG. 2 is a diagram schematically showing an example of the second embodiment of the present invention. As shown in (a), the organic crystal 1 is picked up by the loop-shaped holder 2 together with the culture solution 3 used for the growth, and is held in the holder 2 by the surface tension of the culture solution 3. At that time, the organic crystals 4 other than the object to be measured are picked up at the same time and enter the evaluation region. Even if the characteristic evaluation is performed in such a state, the obtained result is obtained by stacking information from the organic crystal 1 and the organic crystal 4, so that accurate evaluation may not be possible. This is the same whether the organic crystal 4 is the same organic crystal as the organic crystal 1 to be measured, a different organic crystal, or even a foreign substance.

  In this state, the organic crystals 1 and 4 and the culture solution are frozen, and the organic crystals 4 other than the measurement target are removed by irradiation with ultraviolet light (b). In this case, ultraviolet light is irradiated along the vertical cutting line, and the holder 2 and the culture solution 3 on the rear side of the organic crystal 1 are also removed together with the organic crystal 4. On the other hand, the holder 2 and the culture medium 3 on the front side of the organic crystal 1 are left without being removed. In that sense, the first embodiment provides a more accurate evaluation result, but this is sufficient when the holder 2 and the culture solution 3 have little influence on the measurement.

  In the second embodiment, the organic crystal 4 other than the measurement target is removed by cutting the holder 2 and the culture solution 3. However, by irradiating the organic crystal 4 with ultraviolet light, The crystal 4 may be modified or refined so as not to affect the characteristic evaluation.

  In particular, when the organic crystal 1 is a biopolymer crystal, liquids containing glycerol, ethylene glycol, polyethylene glycol, methylpentanediol, paraffin oil, paraton oil, and the like are often used as the culture solution 3. In any case, these culture solutions can be removed by irradiating with appropriate ultraviolet light. The culture medium 3 is not limited to the above-described substances, and any technique described in this specification can be applied as long as the ultraviolet light to be irradiated is absorbed.

  The X-ray crystal structure analysis of the protein crystal which is one of the organic crystals was performed by applying the method for producing the sample for property evaluation in the present invention. The grown protein crystals (egg white lysozyme crystals) are picked up by a holder together with a culture medium consisting of palaton oil and removed by irradiation with an ultraviolet laser along the circumference of the egg white lysozyme crystals in a state of being frozen at −150 ° C. or lower. Then, substances in the X-ray irradiation region were removed, and the X-ray diffraction results before and after the removal were compared.

As a light source of the irradiation apparatus for removal, a short pulse ultraviolet laser by the eighth harmonic generation of a fiber light source having a wavelength of 1547 nm was used. The wavelength of the output light was 193 nm and the pulse width was 1 ns. After passing through the mechanical shutter, the ultraviolet light was reflected by two optical galvanometer mirrors, and the sample was condensed and irradiated by a synthetic quartz lens. The light density on the irradiated surface was 50 mJ / cm ² , the spot diameter was 20 μm, and the laser irradiation position was changed by rotating the galvanometer mirror.

  The result of X-ray crystal structure analysis is shown in FIG. 3 together with a crystal photograph. In FIG. 3, “before” written in the photograph shows a state in which egg white lysozyme crystals are picked up with a holder and frozen. What is written “After (1)” indicates a state in which the periphery of the egg white lysozyme crystal is being removed by irradiation with ultraviolet laser light. What is written “After (2)” is obtained by removing the culture solution around the egg white lysozyme crystal and processing the crystal into a spherical shape, and what is written “After (3)” An additional process is performed on the crystal to reduce the remaining crystal part.

  FIG. 3 shows the results of X-ray diffraction performed in the “before” state, the “After (2)” state, and the “After (3)” state. In FIG. 3, what is written on the right side of the photograph is the X-ray generator, detector, temperature, distance to the detector, opening angle, and irradiation time used for X-ray diffraction.

  The X-ray diffraction results are shown on the lower side of the photograph. The “before” state, the “After (2)” state, the Space Group (space group) in “After (3)”, the Cell dimensions (grid constant), Resolution limit (resolution), Total of observations (number of all reflections measured), of unique observation (number of independent reflections), Completeness (data integrity), Rmerge (data accuracy), Estimated mosaicity (crystal mosaicity) and I / σ (diffraction intensity).

  That the lattice constant is not changed by laser processing means that the crystal structure of the measurement object is not changed by processing, which is a desirable result. Further, the smaller the resolution, the better. However, the fact that the value hardly changes by processing means that the crystal structure of the measurement object has not changed by processing, which is a desirable result. Similarly, the number of all measured reflections, the number of independent reflections, and the completeness of the data are almost unchanged due to processing, which means that the crystal structure of the object being measured has not changed due to processing. Is a desirable result.

  On the other hand, the data accuracy and the mosaic value of the crystal are smaller in the “After (2)” state and “After (3)” than in the “before” state. These values indicate that the smaller the value is, the higher the reliability of the analysis result, and the more reliable data was obtained as a result of removing the material around the measurement object by processing. Yes.

  Further, the intensity of diffraction is reduced by laser processing, which is an appropriate result because the size of the crystal to be analyzed is reduced by processing.

  The X-ray crystal structure analysis of the protein crystal was performed by applying the method for producing a sample for property evaluation in the present invention. When two egg white lysozyme crystals are in close contact with each other and are present in the X-ray transmission region with the egg white lysozyme crystal being picked up with a holder, they are not subject to measurement by ultraviolet light irradiation. The measurement was performed after removing egg white lysozyme crystals. The results are shown in FIG. 4 together with a crystal photograph.

  The upper center photograph in FIG. 4 shows the state of the crystals scooped up by the holder, and the state is schematically shown on the upper left side. Using the same ultraviolet light irradiation apparatus as in Example 1, the entire holder was cut vertically to remove egg white lysozyme crystals that were not to be measured. The upper right photo shows the state after removal, the lower left photo shows the state of the crystal before removal, and the lower right photo shows the state of the crystal after removal. As can be seen from the lower photograph, part of the egg white lysozyme crystal to be measured is also removed during processing.

  The X-ray generator data used are shown on the right side of the photograph, and the X-ray diffraction data is shown on the bottom of the photograph. “Twin” indicates data in a state where two organic crystals exist before processing, and “Detwin” indicates data when organic crystals that are not measurement objects are removed by processing.

  In the case of Twin, the diffraction from two crystals overlapped and the structure analysis could not be performed, whereas in the case of Detwin, an X-ray diffraction pattern from one single crystal was obtained. Data analysis can be performed without problems.

Claims (2)

A method for preparing a sample used for evaluating characteristics of an organic crystal, comprising: freezing the organic crystal in a culture solution containing the organic crystal, preparing a culture solution containing the organic crystal, and containing the organic crystal The culture solution is held by a holding means , and at least a part of the substance other than the evaluation target existing in the vicinity of the organic crystal to be evaluated is removed, modified, or refined by irradiating with ultraviolet light. A method for producing a characteristic crystal characteristic evaluation sample. The characteristic evaluation of the organic crystal is a characteristic evaluation of the surface or internal state of the organic crystal using a probe that is brought into contact with or close to the organic crystal, and is held before and after the organic crystal by irradiating the ultraviolet light. 2. The method for producing a crystal property evaluation sample according to claim 1 , wherein the surface of the organic crystal is exposed by removing the means and the culture solution .

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