BIOPOLYMER CONCENTRATION METHOD, CRYSTALLIZATION METHOD, AND NANOSTRUCTURED SUBSTRATE

Abstract

Electromagnetic waves are uniformly distributed on the light-receiving surface side by taking advantage of their property of being easily concentrated in sharp parts, and the front area (S.sub.A) on the emission surface side is made larger than the back area (S.sub.B) on the light-receiving surface side (S.sub.A/S.sub.B>1), thereby forming a more moderate electric field region. A reduced gold fine particle group (average particle size: 20 nm) was self-assembled on a transparent polyester resin film and half-submerged and fixed. This base material was repeatedly immersed in an electroless gold plating solution so that gold particles were deposited on the gold fine particles. 10 microliters of a protein solution was added dropwise to this nanostructured substrate, and crystallized by a hanging drop vapor diffusion method.

Claims

1. A biopolymer concentration method for concentrating a biopolymer by impregnating a nanostructured substrate with a biopolymer-containing solution while applying electromagnetic waves, wherein the nanostructured substrate comprises a base material, a base group fixed to the base material, and a metal layer group deposited on the base group, the base group is fixed separately to the base material, and a ratio (S.sub.A/S.sub.B) of a geometric front area (S.sub.A) in terms of hemisphere on a total emission surface side of the metal layer group and a geometric back area (S.sub.B) on a total light-receiving surface side exceeds 1.

2. A biopolymer crystallization method for crystallizing a biopolymer by impregnating a nanostructured substrate with a biopolymer-containing solution while applying electromagnetic waves, wherein the nanostructured substrate comprises a base material, a base group fixed to the base material, and a metal layer group deposited on the base group, the base group is fixed separately to the base material, and a ratio (S.sub.A/S.sub.B) of a geometric front area (S.sub.A) in terms of hemisphere on a total emission surface side of the metal layer group and a geometric back area (S.sub.B) on a total light-receiving surface side exceeds 1.

3. The biopolymer concentration method or crystallization method according to claim 1, wherein the metal layer has a peak-valley structure.

4. The biopolymer concentration method or crystallization method according to claim 1, wherein the base group is a metal fine particle group.

5. The biopolymer concentration method or crystallization method according to claim 1, wherein the metal layer group is composed of a reductively deposited metal or alloy.

6. The biopolymer concentration method or crystallization method according to claim 1, wherein the metal layer group, or the metal layer group and the base group show plasmon characteristics.

7. The biopolymer concentration method or crystallization method according to claim 1, wherein the biopolymer is a membrane protein.

8. A nanostructured substrate for biopolymer concentration or crystallization to be irradiated with electromagnetic waves, wherein the nanostructured substrate comprises a base material, a base group fixed to base material, and a metal layer group deposited on the base group, the base group is fixed separately to the base material, and a ratio (S.sub.A/S.sub.B) of a geometric front area (S.sub.A) in terms of hemisphere on a total emission surface side of the metal layer group and a geometric back area (S.sub.B) on a total light-receiving surface side exceeds 1.

9. The nanostructured substrate for biopolymer concentration or crystallization according to claim 8, wherein the metal layer has a peak-valley structure.

10. The nanostructured substrate for biopolymer concentration or crystallization according to claim 8, wherein the base group is a metal fine particle group.

11. The nanostructured substrate for biopolymer concentration or crystallization according to claim 8, wherein the metal layer group, or the metal layer group and the base group show plasmon characteristics.

12. The nanostructured substrate for biopolymer concentration or crystallization according to claim 8, wherein the base material is a resin film with an absorbance of 0.05 or more.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0094] FIG. 1 shows a principle of protein crystallization, and FIGS. 1(a)-1(f) are conceptual diagrams for explaining the principle of the present invention.

[0095] FIG. 2 is a view showing the fine particle group of a comparative example.

[0096] FIG. 3 is a view showing an example.

[0097] FIG. 4 is a view showing an example.

[0098] FIG. 5 is a view showing an example.

[0099] FIG. 6 is a view showing the electric field polarizations of the examples and comparative example.

[0100] FIG. 7 is a conceptual diagram for explaining the examples.

[0101] FIG. 8 is a micrograph of the example.

[0102] FIG. 9 is a micrograph of the example.

[0103] FIG. 10 is a micrograph of an example.

[0104] FIG. 11 is a micrograph of an example, and FIGS. 11(a) and 11(b) are conceptual diagrams of membrane protein.

[0105] FIG. 12 is a conceptual diagram for explaining a conventional example.

[0106] FIG. 13 is a conceptual diagram for explaining a conventional example.

[0107] FIGS. 14(a) and 14(b) are conceptual diagrams for explaining a conventional example.

[0108] Next, the examples of the present invention will be described in detail, together with the comparative example and conventional examples, with reference to the drawings. However, the present invention is not limited to these examples. The metal sheet of the present invention can be achieved with various modifications within the scope of the technical idea of the present invention.

COMPARATIVE EXAMPLE

[0109] A reduced gold fine particle group (average particle size: 20 nm) was self-assembled on a transparent, semi-curable polyester resin film (glass transition temperature (measured value): 140° C., the absorption spectrum curve is the lowest curve in FIG. 6), and the reduced gold fine particle group was half-submerged and fixed by predetermined heat treatment. This is shown in FIG. 2. The absorption spectrum curve is the second curve from the bottom in FIG. 6. The ratio (S.sub.A/S.sub.B) of the geometric front area (S.sub.A) and back area (S.sub.B) is 1. A protein crystallization experiment was carried out in the same manner as in Example 1, described later, except for using this nanostructured substrate. No crystals were deposited even after 7 days passed.

Example 1

[0110] Next, this transparent base material was immersed in an electroless gold plating solution (an improved bath of Preciousfab ACG3000WX, produced by Electroplating Engineers of Japan Ltd.) at 60° C. for 15 seconds, which was taken as 1 cycle. This step was repeated for 6 cycles to obtain a gold metal layer. Specifically, this is a composite particle group in which gold particles are deposited on the fixed gold fine particles. This is shown in FIG. 4. It is obvious that the geometric front area (S.sub.A) in terms of hemisphere on the total emission surface side of the metal layer group shown in FIG. 4 is larger than the geometric back area (S.sub.B) on the total light-receiving surface side.

[0111] As is clear from FIG. 4, an enormous number of peak-valley structures are observed. L-shaped block structures are formed in various places of the peak area. The L-shaped block still shows a peak-valley structure of multiple composite particles. The absorption spectral distribution of the nanostructured substrate was examined. This absorption spectrum curve is shown as the second curve of the example from the top in FIG. 6. As for this diameter-increasing effect of Example 1, the peak value of plasmon due to the horizontal electric field polarization is red-shifted from around 530 nm to around 580 nm. In other words, this shift indicates that the apparent aspect ratio increases. Further, the plasmon due to the vertical electric field polarization is observed at around 870 nm on the right side of the curve. This plasmon peak curve is similar to the plasmon peak curve of a nanorod.

<Protein Crystallization>

[0112] A chicken egg white lysozyme was used as the protein. The protein concentration was 15 mg/mL, and a NaCl 0.5/M solution was prepared as a precipitant. The degree of supersaturation of this solution is 1.25, and it is a metastable solution in which spontaneous crystallization does not occur despite the supersaturation.

[0113] Crystallization was performed by a hanging drop vapor diffusion method, as shown in FIG. 7. 10 microliters of a protein solution was added dropwise to the peak-valley structures of the nanostructured substrate shown in FIG. 4. The nanostructured substrate was turned over, and the chamber was sealed to prevent the evaporation of the protein solution. The chamber was filled with a reservoir solution containing sodium chloride at the same concentration as that of the dropped protein solution. After 1-hour irradiation with light of a xenon lamp through a cut-off filter for cutting a wavelength of less than 600 nm, the resultant was allowed to stand in a thermostatic incubator at 20° C.

[0114] When observed one day after the experiment was started, fine crystals appeared, as shown in FIG. 8. The appearance of fine crystals supports the fact that more crystal nuclei were formed. That is, it is revealed that crystallization was promoted through a series of steps of concentrating the protein single-molecules due to the electric field polarization of the gold metal layer reductively deposited on the gold fine particle group, and forming more crystal nuclei.

Example 2

[0115] A protein crystallization experiment was carried out in the same manner as in Example 1, except that the light from the xenon lamp was linearly polarized using a polarizer. In comparison with Example 1, about four times as many crystals appeared. This result reveals that due to the electric field polarization of the nanostructured substrate shown in FIG. 4, the protein adsorbed and aligned on the gold surface was concentrated, and the formation of crystal nuclei proceeded simultaneously over a wide area.

Example 3

[0116] Example 1 was repeated, except that the gold plating step was repeated for 9 cycles to obtain a gold metal layer. This is shown in FIG. 5. The absorption spectrum curve is the uppermost curve in FIG. 6. A crystallization experiment was carried out in the same manner as in Example 1, except for using this nanostructured substrate. It is obvious that the ratio (S.sub.A/S.sub.B) of the geometric front area (S.sub.A) and back area (S.sub.B) exceeds 1. When observed one day after the experiment was started, many fine crystals appeared, as shown in FIG. 9.

Example 4

[0117] A silver metal layer was formed in the same manner as in Example 1, except for using electroless silver plating. A 4-well simultaneous crystallization experiment was carried out in the same manner as in Example 1, except for using this nanostructured substrate. When observed one day after the experiment was started, many fine crystals appeared in one out of the four wells.

Example 5

[0118] As a membrane protein, the highly halophilic bacterium Halobacterium salinarum was cultured to obtain solubilized bacteriorhodopsin that was concentrated to 19 mg/mL. This solution was mixed with monoolein lipid having a water content of 40% w/w to form a cubic phase. As a salt solution, a 3 molar Na/phosphate buffer solution (pH=5.5) was used to adjust the salt concentration to 2.0 M.

[0119] A 4-well simultaneous crystallization experiment was carried out in the same manner as in Example 1, except for using this membrane protein solution. When observed after 28 days, crystals appeared in one out of the four wells. This is shown in FIG. 10.

Example 6

[0120] A 4-well simultaneous crystallization experiment was carried out in the same manner as in Example 1, except that 7 days later, light from a xenon lamp was applied through a cutoff filter for 1 hour. When observed after 14 days, crystals appeared in two out of the four wells. Further, after 28 days, crystals of the membrane protein appeared in three out of the four wells.

[0121] The crystals of the membrane protein after 14 days had the same size as in FIG. 10. The crystal photograph of the membrane protein after 28 days was larger than 50 μm, as shown in FIG. 11 (left micrograph). The micrographs shown in FIGS. 10 and 11 indicate that the crystal size of the membrane protein increases with the number of times of xenon lamp irradiation. That is, FIGS. 10 and 11 show that due to the electric field polarization of the nanostructured substrate shown in FIG. 4, the membrane protein was concentrated (FIG. 11(a)), the cluster formation was promoted (FIG. 11(b)), and the network of crystal nuclei progressed over a wide area.

Example 7

[0122] Example 1 was repeated, except that the gold plating step was repeated for 4 cycles to form a gold metal layer. This is shown in FIG. 3. The absorption spectrum curve is the third curve from the bottom in FIG. 6. It is obvious that the ratio (S.sub.A/S.sub.B) of the geometric front area (S.sub.A) and back area (S.sub.B) exceeds 1. When observed after 7 days, crystals appeared.

[0123] As is clear from the results of Examples 1 to 7 and the comparative example described above, when the nanostructured substrate according to the present invention was impregnated with a biopolymer-containing solution, the biopolymer was crystallized. It is also found that biopolymer crystals are deposited in the nanostructured substrate according to the present invention by irradiation of electromagnetic waves. This indicates that biopolymer clusters are formed at many sites by electric field polarization, and that these crystal nuclei constitute a planar network and are crystallized. It can be easily understood that this biopolymer crystallization effect can be further enhanced by optimizing the irradiation conditions.

INDUSTRIAL APPLICABILITY

[0124] The biopolymer concentration method or crystallization method of the present invention is effective for the crystal growth of biopolymers. Further, the biopolymer concentration and crystal growth device of the present invention can be used for the detection of environmental hazardous substances, viruses, and the like. Moreover, the biopolymer concentration and crystal growth method etc. of the present invention are available for the industry of chemical and biological measurement, such as chemical sensors and biosensors.