JP4402646B2 - Particle, sensor using particle, and method for producing porous structure unit - Google Patents

Description

  The present invention relates to particles having biological material held in mesopores. In particular, by using this particle to selectively detect a specific biological substance, it can be applied to a sensor for diagnosing diseases such as cancer.

  Technology for immobilizing biomaterials, particularly biomolecules, on insoluble carriers can be applied to biocatalysts for bioproduction, detection devices for biomaterials, and the like, and thus technology is currently being actively developed. In particular, a technique for performing an antigen-antibody reaction based on a high-level molecular recognition reaction outside a cell is a very important technique for diagnosis of diseases and the like. However, in many cases, the three-dimensional structure of a protein is directly related to the expression of a function, and particularly, an intracellular protein tends to undergo a three-dimensional structure change outside the cell. As a result, the function expressed in the living body is often lost. This is a major challenge for the development of devices using proteins, and the technology to stably immobilize them on a carrier while maintaining the activity of the protein and thus the three-dimensional structure is very important. is there.

  As one of the techniques for immobilizing proteins, there is a technique that uses a fine space of a porous material. Examples of this technique include those using an inorganic material produced by a sol-gel method, mesoporous silica, a porous organic polymer, porous silicon, porous glass, and the like. Furthermore, regarding mesoporous silica, Patent Document 1 discloses a technique for supporting an antibody, and Patent Document 2 discloses a technique for immobilizing an enzyme.

  On the other hand, Non-Patent Document 1 discloses a technique for synthesizing mesoporous silica fine particles having a relatively uniform particle size and a very small particle size by combining a nonionic surfactant and a cationic surfactant. Is disclosed.

  However, these prior arts have some improvements as follows.

  That is, the porous organic polymer may not have sufficient mechanical strength. Moreover, in the case of porous silicon, it is opaque and it is difficult to optically confirm the immobilization of biomolecules.

  In this respect, mesoporous silica can be an excellent host material, but in this case, problems remain in the pore diameter and its arrangement. First, regarding the pore diameter, the pore diameter of mesoporous silica is often too small compared to the size of the biomaterial. Regarding the arrangement of the pores, in the case of tube-shaped pores, it is difficult to increase the amount of biological material immobilized because there are few cross-sections in which the openings of the pores are exposed. In the case of a three-dimensional pore such as a cubic structure, the size of the window connecting the spherical mesopores is small, and depending on the diameter of the substance, it is disadvantageous for diffusing into the mesopores.

For this reason, there has been a demand for a porous material that has both strength, stability, and transparency, and has a large immobilization amount per unit volume of a substance to be immobilized such as a biological substance.
JP 2004-83501 A JP 2000-139459 A Suzuki et al., "Synthesis of Silica Nanoparticulates having a well-ordered mass structure using a double effluent US, Journal of the United States", Journal of the United States. 462-463

  The present invention has been made in view of the above-described problems, and an object of the present invention is to provide particles having a large amount of immobilization per unit volume of a substance to be immobilized, a method for producing the same, and a sensor using the particles. .

  The particles according to the present invention are particles having mesopores holding a biological substance, and the average particle size of the particles is within 10 times the average pore size of the mesopores.

  A sensor according to the present invention is a sensor for detecting a detection substance, and detects binding between the particle according to any one of claims 1 to 5, the detection substance, and the biological substance. And a detecting unit for the purpose.

  The method for producing particles according to the present invention comprises a step of preparing a liquid containing a cationic surfactant, a nonionic surfactant, and a hydrophobic substance that expands micelles, and a silica source is added to the liquid. Forming a silica mesostructure containing the cationic surfactant, the nonionic surfactant, and the hydrophobic substance, the cationic surfactant, the nonionic surfactant, The method comprises removing the hydrophobic substance to form mesopores in the silica mesostructure, and attaching a biological substance to the hollow structure.

  According to the present invention, the opening area per unit volume is increased, which makes it possible to provide particles with a large amount of immobilization per unit volume of the substance to be immobilized, improving sensitivity applicable to disease diagnosis such as cancer. This makes it possible to provide a sensor.

  Hereinafter, the present invention will be described in detail.

  FIG. 1 is a diagram schematically showing the material of the present invention.

  First, the porous material used in the present invention will be described. In the present invention, the porous material is not particularly limited, and examples thereof include silica, titanium oxide, and tin oxide. Hereinafter, the case where silica is used as the porous material will be exemplified. The porous silica particles 11 used in an embodiment of the present invention are mesoporous silica particles produced using a surfactant aggregate as a template, and have pores 12 having a substantially uniform diameter. In FIG. 1, a porous body having a structure in which tube-shaped pores are packed in a honeycomb pack is illustrated, but the structure of the pores is not limited to this structure. For example, a porous body having various pore structures such as a structure in which spherical pores are densely packed three-dimensionally or a structure having a double gyroid structure is applicable to this porous body.

  The pore diameter of the mesoporous silica to be used is not particularly limited, but when it is smaller than the size of a substance to be immobilized such as a biological substance immobilized in the pore, it is difficult to introduce into the pore. For this reason, it is necessary to adjust a pore diameter so that it may become an optimal value with respect to the biological material to be used. The pore diameter of mesoporous silica produced using a cationic (cationic) surfactant is generally about 2 to 3 nm, and this range is smaller than many dimensions of the biological material to be immobilized. . In such a case, it is necessary to increase the pore diameter by adding an additive having an action of swelling micelles to the reaction solution. Trimethylbenzene, decane, and linear amines have been reported as substances having the effect of swelling micelles. Needless to say, any substance capable of swelling micelles can be used. In addition, the method of measuring the isothermal adsorption line of gas, such as nitrogen, can generally be used for evaluation of the pore diameter distribution in the mesoporous silica used for this invention. From the obtained isothermal adsorption line, it is calculated by an analysis method such as Berret-Joyner-Halenda (BJH).

  The shape of the primary particles of the present invention is depicted as a hexagonal plate shape in FIG. 1, but the shape of the primary particles does not have a significant meaning for the material of the present invention. Even shapes can be used. For example, there are spherical shapes, cube shapes, and the like.

  In the present invention, a substance to be immobilized such as a biological substance is immobilized in the pores, and a biological material that forms a selective bond with the substance is detected. In this case, since the amount of the biomaterial to be detected is often a very small amount, it is necessary to immobilize the biomaterial on the porous silica at a high density in order to detect with high sensitivity. For this reason, the specific surface area of the porous silica used as the carrier becomes a problem. However, when the size of the biological material to be immobilized is large as in the present invention, diffusion in the pores becomes a problem. The aspect ratio of the pores and the ratio of the opening area to the volume of the particles are problematic. In the present invention, good results are obtained in the case of porous particles having a fine particle diameter within 10 times the pore diameter. In the present invention, the “biological substance” means an antibody or antigen, as well as various proteins and genes, and an artificial substance combining these. Specific examples include antibodies, various antibody fragments, DNA, RNA, proteins, enzymes, and the like. Biological materials also include single-stranded or double-stranded DNA or RNA, fragments containing active sites such as Fab antibodies, and the like. The DNA fragment may be extracted from animals or plants or microorganisms and cut into a desired shape / length, or may be synthesized / modified by genetic engineering or chemicals.

  Here, a technique for controlling the particle size of mesoporous silica will be described. As this method, the method described in Non-Patent Document 1 can be used. This method is characterized by using a mixture of a cationic surfactant and a nonionic block copolymer surfactant. The cationic surfactant forms micelles in the silica mesostructure and functions as a template for pores of mesoporous silica. On the other hand, the nonionic block copolymer surfactant is considered to have a function of suppressing the growth of the formed silica mesostructured particles. Examples of the cationic surfactant include cetyltrimethylammonium and stearyltrimethylammonium. Preferred examples of the nonionic block polymer surfactant include polyethylene oxide-polypropylene oxide-polyethylene oxide triblock polymer surfactants. However, the above-mentioned cationic and / or nonionic surfactant is not limited to these, and any surfactant may be used as long as the object of the present invention can be achieved.

  The mesoporous silica particles used in the present invention are basically produced using this method. However, as described above, the pores formed by the cationic surfactant are too small to immobilize immobilization substances such as biological substances, so add a substance that works to swell micelles such as trimethylbenzene. To do. Thereby, fine particle mesoporous silica with a large pore diameter can be produced. The pore size can also be increased by aging the formed particles at high temperatures.

  In the present invention, there is another advantage of using primary particles having a small particle size. When fine particles aggregate as shown in FIG. 1, a gap 13 is formed between the particles. In the case of primary particles of the size used in the present invention, the gap 13 formed between the particles is a fine space of less than 50 nm. When the biological material is immobilized in the mesopores, the immobilized material such as the biological material diffuses in this space, reaches the mesopores, and is immobilized. In addition, the pores of this size stabilize the biomaterial to be detected in an antigen-antibody reaction or the like using an antibody or antigen as an immobilized substance and an antigen-containing material as the biomaterial to be detected, as described later. It also plays a role. As a result, this contributes to detecting the detection target with high sensitivity.

  Next, a process for immobilizing a substance to be immobilized such as a biological substance on mesoporous silica will be described.

  What is immobilized in the pores is a substance to be immobilized such as a biological substance, and uses a substance that forms a selective bond with the biological material to be detected. The substance to be immobilized such as a biological substance to be immobilized is not particularly limited, but the antibody or antibody fragment may be immobilized and used to detect a specific antigen (biological material). Conversely, an antigen or antigen fragment may be immobilized and used to detect a specific antibody (biological material). Furthermore, not only antigens / antibodies, but also substances that can form specific bonds between specific molecular species, such as biotin-avidin bonds and genes complementary to specific gene sequences (probes) are immobilized. The substance and / or the detection target may be used.

  There are several methods for immobilizing a substance to be immobilized such as the biological substance 14 in the pores. Even if no particular treatment is performed on the mesoporous silica, the biological material may be taken into the pores and immobilized by simply contacting the mesoporous silica with a solution containing the target biological material (FIG. 1A). )). In addition, the surface of the porous silica may be modified with a silane coupling agent or the like to form a specific functional group R in the pores, thereby improving the immobilization rate and stability (FIG. 1 (B)). . In the present invention, the method for immobilizing the biological material in the pores is not particularly limited.

  However, when the portion to be immobilized is limited to a portion that selectively binds to a biological material to be detected, such as a biological material (antibody or antibody fragment) 15, immobilization of the biological material in the pores. The direction to be converted becomes a problem. In this case, it is preferable to introduce in advance a substance 16 corresponding to a scaffold for immobilizing the substance (biological substance) to be immobilized in the pores. For example, by introducing metallic gold into the pores and bringing it into contact with an artificial antibody having an affinity site with metallic gold at one end, the orientation can be controlled and supported in the pores. (FIG. 1C).

  Next, sensing of a biological substance using the material of the present invention will be described with reference to FIG.

  First, a case where an avidin-biotin recognition reaction is performed will be described as an example. After modifying the pore surface of the fine particle mesoporous silica described above, a site in which biotin is immobilized on the pore surface is formed by chemical bonding. Next, the mesoporous silica fine particles subjected to this treatment are transferred to a container 21 provided with a polyethylene filter 22 having fine pores having a pore diameter of 0.02 μm at the outlet. Here, a dilute solution of streptavidin combined with a fluorescent dye that functions as a fluorescent probe is introduced into the container 21. After an appropriate contact time, the buffer solution is used to filter out excess streptavidin so that only the mesoporous silica particles and the material bound thereto remain on the filter. When this filter is observed with a fluorescence microscope to confirm the fluorescence, the detection of streptavidin is achieved using mesoporous silica particles with biotin immobilized thereon.

  Next, a case where an antigen-antibody reaction is performed will be described.

  The configuration in this case can also be basically detected with the same configuration as the biotin-avidin reaction described above. First, an antibody or antibody fragment is immobilized on the pore surface of the above-mentioned mesoporous silica. Specifically, the particles are transferred to a container 21 provided with a polyethylene filter 22 having fine pores having a pore diameter of 0.02 μm at the outlet, and a solution containing a minute amount of an antibody (biological substance) is introduced into the container 21. . After an appropriate contact time, excess antibody is filtered off using a buffer solution. Next, an antigen (biological material) to which a fluorescent marker is bound in advance is introduced into the container 21 and brought into contact with each other for an appropriate time, and then washed again with a buffer solution, and excess antigen is removed by filtration. By this operation, only the mesoporous silica particles and the substance bonded thereto remain on the filter. When this filter is observed with a fluorescence microscope and fluorescence is confirmed, a specific antigen-antibody reaction can be confirmed on the mesoporous silica particles.

  When immobilizing an antibody, the direction of immobilization of the antibody with respect to the opening of the pore becomes a problem. Therefore, in such a case, if a material that functions as a foothold for antibody binding is first formed on the porous body, and the antibody is bound to that material, a favorable result can still be obtained. It is done.

  The present invention includes a biological detection means (biosensing device) having a reaction system including a reaction field having the functions as described above, and a detection system for finally detecting the presence of a substance to be detected. . In this case, the reaction system may be any system as long as it can perform the above-described series of operations, and the detection system may be any system that can detect the target biomaterial in a minute amount. It may be a means, and is not limited to the measurement of fluorescence.

  The gist of the present invention described above makes it possible to immobilize a biological material having a relatively large size using porous particles having a fine particle diameter within 10 times the pore diameter. As a result, the amount of immobilization per unit volume can be increased, and as a result, a biosensing element capable of detecting a selective reaction of a biological substance with high sensitivity is produced.

  EXAMPLES Hereinafter, although this invention is demonstrated in more detail using an Example, this invention is not limited to the content of an Example.

Example 1
In this example, cetyltrimethylammonium chloride is used as a cationic surfactant, Pluronic F127 triblock copolymer (BASF) is used as a nonionic surfactant, and trimethylbenzene is used as a substance that swells micelles. In this example, biotin is bound to the prepared mesoporous silica particles, and streptavidin is detected with high sensitivity by fluorescence measurement.

  26.0 g of cetyltrimethylammonium chloride and 20.0 g of F127 were dissolved in 300 g of hydrochloric acid adjusted to pH = 0.5 in advance, and 11.0 g of N, N, -dimethylhexadecylamine was added and stirred for 4 hours. . To this, 35.0 g of tetraethoxysilane as a silica source was added and stirred at room temperature for 24 hours. To this, 30.0 g of 15M concentrated aqueous ammonia was added, and the mixture was further stirred for 24 hours. This was dried at room temperature under vacuum for 24 hours and then calcined at 540 ° C. for 10 hours to remove the surfactant. In this way, mesoporous silica particles were obtained.

The sample after baking was evaluated using a BET gas adsorption device. As a result, the mesoporous silica particles described above had an average pore size distribution of 5.5 nm and a specific surface area of 1100 m ² / g. By observing this sample with a transmission electron microscope, it was found that the average particle size of the powder had a uniform particle size of 50 nm.

  Next, the fired particles were treated with aminopropyltriethoxysilane to bind amino groups to the surface. Following this, the particles were dispersed in a 15 mM DMF solution of biotin-N-hydroxy-succinimide ester and sonicated for 10 minutes. After separating the particles, they were thoroughly washed with DMF and ultrapure water in this order, and dried under vacuum at room temperature.

  10 mg of these particles were transferred to a container 21 having an inner diameter of 3 mm and provided with a filter 22 having a pore diameter of 0.02 μm at the outlet, as shown in FIG. 2, and a 2.5 μM solution of streptavidin labeled with Cy5 therein. Was introduced. At this time, 0.01M Phosphate buffered saline was used as a solvent.

  After introducing this solution and allowing to stand at room temperature for 15 minutes, excess streptavidin was removed by filtration with a filter, and further washed well with 0.01 M Phosphate buffered saline.

  Finally, the powder after these treatments was observed with a fluorescence microscope, and clear fluorescence was confirmed. From these facts, it is clear that streptavidin in a solution can be detected with high sensitivity using the biotin-avidin selection reaction using the particles of the present invention.

(Example 2)
Using the same reagent as in Example 1, mesoporous silica fine particles were produced under the same conditions.

  The mesoporous silica fine particles were treated with an N-trimethoxypropyl-N, N, N-trimethylammonium chloride solution and thoroughly washed with ethanol.

  After drying this, it was immersed in a tetrachloroauric (III) acid saturated aqueous solution, separated, washed, and heated at 200 ° C. in a hydrogen gas atmosphere. When this was observed with a transmission electron microscope, it was confirmed that gold derived from the tetrachlorogold (III) acid saturated aqueous solution was formed in the pores.

  Subsequently, the mesoporous silica particles carrying metal gold were brought into contact with a buffer solution of an artificial antibody having a gold-affinity site to immobilize the artificial antibody on the gold. As schematically shown in FIG. 3, this artificial antibody is composed of a part 32 that selectively recognizes gold and a part 31 that recognizes egg white lysozyme (HEL), and these are single-chain Fv. It was a connected structure. Each recognition site was a variable region domain of a HEL antibody and a variable region domain of an antibody that recognizes gold.

  Antibodies having a strong affinity for gold were selected by screening using the phage display method. The artificial antibody used in this example was prepared by genetic engineering from this antibody having a strong affinity for gold and an anti-HEL antibody.

  10 mg of the above mesoporous silica particles supporting metal gold was transferred to a container 21 having an inner diameter of 3 mm and a pore diameter of 0.02 μm at the outlet as shown in FIG. When 1 mL of 0.01M Phosphate buffered saline solution of this artificial antibody was introduced there, the artificial antibody was bound to the gold fine particles at the gold recognition site 32, and the HEL recognition site 31 was directed to the opening of the pore. In this state, after maintaining at room temperature for 1 hour, 0.01M Phosphate buffered saline solution was flowed, and excess artificial antibody was removed by filtration.

  A 1 μM HEL solution was introduced into the mesoporous silica supporting the metallic gold and the artificial antibody, and HEL was further bound thereto.

  Further, a 0.01 μM phosphate buffered saline solution of 1 μM anti-HEL antibody was introduced into this, and after holding for 1 hour, it was thoroughly washed with 0.01M phosphate buffered saline.

  Thereafter, 10 μM of 0.01 M Phosphate buffered saline solution of anti-IgG antibody bound with rhodamine as a label was introduced. Then, after holding again for 1 hour, it was thoroughly washed with 0.01M Phosphate buffered saline.

  After the operation, the powder remaining on the filter was dried and then observed with a fluorescence microscope, and fluorescence from rhodamine was confirmed. Thus, the antigen-antibody reaction can be monitored with high sensitivity using the mesoporous silica of the present invention as a reaction carrier.

(Example 3)
In this example, the same biotin-avidin reaction as in Example 1 was detected using a biological detection means (biosensing device) provided with fluorescence detection means and sample pretreatment means.

  The living body detection means of the present invention is roughly divided into a reaction part and a detection part.

  First, the reaction unit has a vacuum container 45 that holds the container 21 shown in FIG. 4 and is connected to a vacuum pump 47 through an exhaust port 46. A solution is introduced into the mesoporous silica powder held on the filter 22 through a tube from a buffer solution container 41, a solution container 42 containing a biological material, and a solution container 43 containing a biological material. . The tube was provided with a valve 44 for controlling the introduction amount of various solutions. By reducing the pressure in the vacuum container 45, the solution accumulated on the filter 22 was filtered by the filter 22, collected in the vacuum container 45, and then discharged from the exhaust port 48 to the waste liquid container 49.

  The powder prepared in Example 1, treated with aminopropyltriethoxysilane, then treated with biotin-N-hydroxy-succinimide ester, washed and dried was placed on the filter 22.

  A 2.5 μM solution of streptavidin labeled with Cy5 was placed in the container 43, and the valve was opened and introduced onto the filter. Since the filter has very small pores, the solution was retained on the filter unless the inside of the container was depressurized. In the same manner as in Example 1, after maintaining for 15 minutes, the vacuum pump was operated, the inside of the container was depressurized, and the excess streptavidin solution was removed by filtration. Thereafter, a buffer solution was introduced from the container 41 while the inside of the vacuum container was kept under reduced pressure, and sufficient washing was performed. After washing, the vacuum pump was stopped and the inside of the container was returned to normal pressure. Then, the container 21 was taken out, and the filter 22 to which mesoporous silica was adhered was removed.

  In the detection unit described below, excitation light having a wavelength selected by the incident light unit was applied to the filter that had been processed in the reaction unit, and a fluorescence spectrum was recorded in the detection system. In order to detect weak light, the detection system has a structure that blocks light, and in some cases, a means for cooling the detector is provided separately.

  FIG. 5 is a diagram schematically illustrating the above-described detection unit. The detection unit basically has the same configuration as that of a normal fluorescence measurement device. That is, it has an incident light unit 51 including a light source and a spectroscope, a jig 52 for fixing the filter with the mesoporous silica fine particles, and a detection unit 54 including a spectroscope and a detector. The optical unit 53 includes a mirror that guides light to the filter and guides light to the detection unit.

  As a result of carrying out the reaction of Example 1 using this apparatus, a clear fluorescence spectrum was observed, and the biotin-avidin selective binding reaction could be monitored well.

Example 4
In this example, the same biotin-avidin reaction as in Example 2 was detected using a biological detection means (biosensing device) having basically the same configuration as in Example 3.

  The mesoporous silica particles carrying metal gold produced in Example 2 were placed on the filter 22 of the apparatus shown in FIG.

  The buffer solution of the artificial antibody having the gold affinity site and the HEL affinity site described in Example 2 was placed in the container 42, and the valve was opened and introduced onto the filter. Since the filter has very small pores, the solution was retained on the filter unless the inside of the container was depressurized. After maintaining in this state for 15 minutes, the vacuum pump was operated to reduce the pressure in the container, and excess artificial antibody was removed by filtration. Thereafter, a buffer solution was introduced from the container 41 while the inside of the vacuum container was kept under reduced pressure, and sufficient washing was performed.

  After washing, the vacuum pump was stopped and the inside of the container was returned to normal pressure. Then, 1 μM anti-HEL solution placed in the container 42 was introduced onto the filter and held for 1 hour. Then, the vacuum pump was operated again, and excess HEL was sufficiently washed with the buffer solution from the container 41.

  After stopping the vacuum pump, 1 μM anti-HEL antibody / 0.01 M Phosphate buffered saline solution is put into the container 42, the valve is opened, and the solution is introduced onto the filter 22 and held for 1 hour. Thereafter, the vacuum pump was operated again, and the plate was thoroughly washed with 0.01M Phosphate buffered saline.

  Finally, 0.01 M Phosphate buffered saline solution (10 μM) of anti-IgG antibody bound with rhodamine as a label was placed in the container 43, introduced onto the filter 22, and held again for 1 hour. Then, it was washed well with 0.01M Phosphate buffered saline.

  After washing, the vacuum pump was stopped and the inside of the container was returned to normal pressure. Then, the container 21 was taken out, and the filter 22 to which mesoporous silica was adhered was removed.

  This filter was fixed to the sample fixing holder of the detection unit having the same configuration as that of Example 3, the fluorescence spectrum was measured, and the fluorescence spectrum derived from rhodamine was confirmed.

  As described above, although the number of containers in which the solution is put is different from that in Example 3, the target antigen-antibody reaction can be monitored well as a result of performing the reaction in Example 2 using the same apparatus. , Antigens can be detected.

  The present invention has the effects as described above, and is expected to be widely applicable to biocatalysts, biomaterial detection devices, and the like.

It is a schematic diagram regarding the mesoporous silica which comprises the reaction field for biological substance selective reaction of this invention. In this invention, it is a schematic diagram of the reaction container for hold | maintaining mesoporous silica and performing reaction of a biological substance in vitro. It is the figure which showed typically the artificial antibody produced in Example 2 of this invention. It is a schematic diagram of the reaction part of the biosensing device used in Example 3 of the present invention. It is a schematic diagram of the detection part of the biosensing device used in Example 3 of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Porous silica particle 12 Pore 13 Crevice 14 Biological substance 15 Biological substance 16 Substance 21 Container 22 Polyethylene filter 31 Part 32 Part 41 Container 42 Container 43 Container 44 Valve 45 Vacuum container 46 Exhaust port 47 Vacuum pump 48 Exhaust port 49 Waste liquid container 51 Incident Light Unit 52 Jig 53 Optical Unit 54 Detection Unit

Claims (1)

And cationic surfactants, and nonionic surfactant, preparing a liquid containing a substance swelling the micelles,
Adding a silica source to the liquid to form a plurality of silica meso fine particles including the cationic surfactant, the nonionic surfactant, and the hydrophobic substance;
Aggregating the plurality of silica meso fine particles to form fine voids of less than 50 nm between the silica meso fine particles;
The cationic surfactant, the nonionic surfactant, and the hydrophobic substance are removed to form mesopores in the silica mesoparticles , and the average particle size of the silica mesoparticles is determined as the average pore size of the mesopores. A step of 10 times or less ,
In the mesopores, method for producing fine particles, which comprises a step of attaching the biological material.

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