KR100917465B1 - A method of separating a microorganism using a nonplanar solid substrate and device for separating a microorganism using the same - Google Patents

Abstract

The present invention provides a method for separating microorganisms, the method comprising contacting a non-planar solid support with a sample containing microorganisms in the range of pH 3.0 to 6.0.

Non-planar shape, solid support, cells, enrichment

Description

A method of separating a microorganism using a nonplanar solid substrate and device for separating a microorganism using the same}

1 is a diagram showing the effect of E. coli attachment to a solid support having a type of buffer present in the flow apparatus and having a columnar array formed thereon.

FIG. 2 shows the effect of salt concentration on the attachment of E. coli to a solid support having a surface present in a flow apparatus and having a columnar array formed thereon.

FIG. 3 shows the effect of pH and the properties of the surface on which the array of pillars are formed in the presence of E. coli in the blood on a solid support that is present in the flow apparatus and has the surface on which the pillar arrays are formed.

FIG. 4 shows a sample of blood diluted with acetate buffer (pH 5.2) flowing through a solid support having a surface present in a flow apparatus and having a columnar array formed therein, and washed with 100 mM sodium acetate (pH 4.0) Then, it is the result of observing the surface in which the said column array is formed with the optical microscope.

5 is a view showing the effect on the solid support adhesion of E. coli in the sample according to the diluted urine sample pH.

FIG. 6 shows the effect of pH and surface properties on the adhesion of E. coli in a diluted urine sample to a solid support having a surface on which a columnar array is formed.

Figure 7 shows the result of separating the cells in the urine using a solid support having a surface on which the columnar array is formed, showing the result of the variation of the urine.

8 shows the result of separating E. coli in urine using a solid support having a surface on which pillar arrays are formed, and shows the result of dilution of urine.

Fig. 9 shows the results of the E. coli separation in urine using a solid support having a surface on which pillar arrays are formed.

FIG. 10 shows the results of concentration of E. coli in urine using a solid support having a surface on which pillar arrays are formed, showing the results of dilution and flow rate of urine.

The present invention relates to a method for separating microorganisms using a non-planar solid support and a microorganism separation apparatus.

In the conventional method of separating microorganisms, centrifugation and filtration processes have been generally used. In addition, in order to concentrate or isolate a specific cell, a method of specifically binding the cell to a support to which a receptor or ligand that specifically binds the specific cell is bound, and a method of concentrating or separating the cell has been used. For example, by flowing a sample containing the cells to a support having an antibody binding to a protein specific to the cells bound to bind the cells to the antibody, and washing the unbound cells Affinity chromatography methods are included.

In addition, Korean Patent Publication No. 2006-0068979 discloses a piezoelectric transducer connected to both ends of an upper glass substrate and converting an electrical input from the outside into mechanical vibration to apply to the upper glass substrate; And N electrodes arranged on a lower substrate parallel to the upper glass substrate, wherein a gap between the upper glass substrate and the lower substrate is filled with a fluid mixed with cells, and each electrode has a length of the piezoelectric transducer. And the N electrodes are arranged in a direction perpendicular to the direction, the cell separation system using an ultrasonic field and traveling wave electrophoresis, characterized in that it comprises a cell separation device arranged at a predetermined interval along the longitudinal direction of the piezoelectric transducer. Is disclosed.

However, all of the prior art as described above is to immobilize a ligand or receptor on a solid substrate, or to provide external power to selectively concentrate or isolate specific cells. Thus, methods and apparatus for separating cells using the properties of the solid support itself and the conditions of the liquid medium are unknown.

It is to provide a method for separating microorganisms using the non-planar solid support of the present invention.

The present invention also provides an apparatus for concentrating a microorganism comprising a non-planar solid support.

The present invention provides a method for separating microorganisms, the method comprising contacting a non-planar solid support with a sample containing microorganisms in the range of pH 3.0 to 6.0.

The present invention includes contacting a non-planar solid support with a sample containing microorganisms in the range of pH 3.0 to 6.0. The contact causes the microorganism to adhere to the solid support.

Among the liquid media in which the solid support is included, the microorganisms may be present in or attached to the solid support. The distribution between this liquid medium and the solid support is known to be determined by the difference between the surface tension of the liquid medium and the surface tension of the bacteria. That is, if the surface tension of the liquid medium is greater than the surface tension of the bacteria, the bacteria adhere better to the solid support, that is, the hydrophobic solid support, the surface tension of the bacteria is greater than the surface tension of the liquid medium, The bacteria adhere better to a solid support having a high surface tension, ie, a hydrophilic solid support, and if the surface tension of the bacteria is equal to the surface tension of the liquid medium, there is no effect of surface tension on the attachment of the solid support of the bacterial cells and an electrostatic Other interactions, such as miracle interactions, have been reported to affect (Applied and Environmental Microbiology, July 1983, p. 90-97). It is also known that through thermodynamic approach based on surface tension, bacterial cells can adhere to surfaces not only by bacterial cells but also through electrostatic attraction characteristics. However, these phenomena have a problem that their attachment speed is very slow.

In order to solve the problems of the prior art as described above, the present inventors use a non-planar solid support, and at the same time by contacting the sample containing microorganisms with the solid support in the range of pH 3.0 to 6.0, It was confirmed that the microorganisms can be separated. This is believed to increase the surface area of the solid support and at the same time, by using a liquid medium of pH 3.0 to 6.0, the cell membrane of the microorganisms is denatured, solubility in the solution is lowered, so that the rate of attachment to the solid surface is relatively high, but of the present invention The scope is not limited to this particular mechanism.

In the contacting step, the sample includes any sample containing a microorganism. Preferably, it is selected from the group consisting of biological samples, clinical samples and laboratory samples containing the microorganisms. In the present invention, "biological sample" means a sample containing or consisting of cells or tissues, such as cells or biological liquids isolated from an individual. The object may be an animal including a human. Examples of biological samples include saliva, sputum, blood, blood cells (e.g. white blood cells, red blood cells), amniotic fluid, serum, semen, bone marrow, tissue or microneedle biopsy samples, urine, peritoneal fluid, pleural fluid and cell culture. Included. Biological samples include tissue sections, such as frozen sections taken for histological purposes. Preferably, the biological sample is a "clinical sample" which is a sample derived from a human patient, and more preferably, the sample is blood, urine, saliva, or sputum.

In the present invention, microorganisms to be isolated include bacterial cells, fungi and viruses.

In the contacting step, the sample may be diluted with a solution or a buffer that can serve as a buffer for the microorganism at low pH. Preferably, the buffer may be diluted with phosphate buffer (eg sodium phosphate, pH 3.0 to 6.0) or acetate buffer (sodium acetate, pH 3.0 to 6.0). The degree of dilution is not particularly limited, but may be preferably diluted at a magnification of 1: 1 to 1: 1,000, more preferably 1: 1 to 1:10.

In the contacting step, the sample is one having a salt concentration of 10 mM to 500 mM, preferably 50 mM to 300 mM. The sample has an ion concentration selected from the group consisting of 10 mM to 500 mM, preferably 50 mM to 300 mM acetate and phosphate.

In the contacting step, the solid support has a non-planar shape and thus has a surface whose surface area is increased compared to the plane. The solid support may be a concave-convex structure is formed on the surface. In the present invention, the "concave-convex structure" refers to a structure having a concave and convex surface without smooth surface. The uneven structure includes a surface having a columnar structure in which a plurality of pillars are formed or a surface having a network structure in which a plurality of pores is formed, but is not limited to these examples.

In the contacting step, the non-planar solid support may have any shape. For example, a solid support having a pillar structure having a plurality of pillars formed on a surface thereof, a solid support having a bead shape, and a sieve structure having a plurality of pores formed on a surface thereof. It may be selected from the group consisting of a solid support. The solid support may be used alone or as a collection of a plurality of the solid supports (eg, an aggregate packed in a tube or a container).

In the contacting step, the solid support may be in the form of an inner wall of the microchannel or microchamber in the microfluidic device. Accordingly, the method for separating microorganisms of the present invention is provided in a fluidic device or microfluidic device in which one or more inlets and outlets are provided and the inlets and outlets are communicated through channels or microchannels. It can be performed in.

One embodiment of the method of the present invention, in the contacting step in the method of the present invention, the solid support may have a columnar structure in which a plurality of pillars are formed on the surface. The manufacture of pillars on solid supports is well known in the art. For example, fine pillars can be formed at high density using a photolithography process or the like used in a semiconductor manufacturing process. The pillar has an aspect ratio of 1: 1 to 20: 1, but is not limited thereto. In the present invention, "aspect ratio" means the ratio of the cross-sectional diameter to the height of the column. The pillar structure may be a ratio of the distance between the pillar height and the pillar is 1: 1 to 25: 1. The pillar structure may have a distance between the pillars in a range of 5 μm to 100 μm.

In the contacting step in the method of the present invention, the non-planar solid support may have a hydrophobicity of 70 ° to 95 ° water contact angle. The hydrophobicity with a water contact angle of 70 ° to 95 ° consists of octadecyldimethyl (3-trimethoxysilyl propyl) ammonium (OTC) and tridecafluorotetrahydrooctyltrimethoxysilane (DFS). The compound selected from the group may be coated and given. The surface having a water contact angle of 70 ° to 95 ° preferably has octadecyldimethyl (3-trimethoxysilyl propyl) ammonium (OTC) and tridecafluorotetrahydrooctyltrimethoxy in the SiO ₂ layer of the solid support. Compounds selected from the group consisting of silanes (DFS) may be self-assembled monolayer (SAM) coated.

In the method of the present invention, in the contacting step, the non-planar solid support may have one or more amine-based functional groups on its surface. The surface having at least one functional group of the amine system may be coated with polyethyleneiminetrimethoxysilane (PEIM) on the surface of the solid support. The coated surface may preferably be a self-assembled monomolecular (SAM) coating of polyethyleneiminetrimethoxysilane (PEIM) on the SiO ₂ layer of the solid support. The amine-based functional group has a positive charge in the range of pH 3.0 to 6.0.

In the method of the present invention, in the contacting step, the solid support includes a support of any material as long as it has water contact angle characteristics as described above or one or more amine-based functional groups on its surface. For example, glass, silicon wafers, plastic materials, and the like are included, but are not limited to these examples. The surface having a water contact angle of 70 ° to 95 ° or a surface having at least one amine-based functional group on the surface thereof is considered to be attached to the microorganism when the sample containing the microorganism contacts the solid support having the surface. The present invention is not limited to a specific mechanism.

The method for separating the microorganism of the present invention may further include washing a substance other than the target microorganism not attached to the solid support after the contacting step. The washing may be any solution capable of removing impurities that may adversely affect subsequent processes without leaving the target microorganisms attached to the solid support surface from the solid support. For example, acetate buffers and phosphate buffers used as binding buffers can be used. The washing solution is preferably a buffer having a pH in the range of pH 3.0 to 6.0.

In the present invention, "separation of microorganisms" includes not only pure separation of microorganisms in a sample but also concentration.

According to the method of the present invention, the microorganisms attached and concentrated on the solid support can be used for further processing steps such as the separation of DNA while attached on the solid support. In addition, the microorganisms attached and concentrated on the solid support can be eluted from the solid support and used for further processing steps.

Thus, the method may further comprise eluting the attached microorganism after the contacting and / or washing step. In the eluting step, any solution known in the art may be used as long as the solution used for eluting has a property of detaching microorganisms from the solid support. For example, water and tris buffers and the like can be used. The elution solution is preferably a solution of pH 6.0 or higher.

The present invention also provides a microorganism separation device including a container including a non-planar solid support, a sample inlet provided in the container, and a buffer solution storage provided in the container.

In the apparatus of the present invention, the solid support has a non-planar shape and has a surface whose surface area is increased compared to the plane. The solid support may be a concave-convex structure is formed on the surface. In the present invention, the "concave-convex structure" refers to a structure having a concave and convex surface without smooth surface. Such uneven structure includes a surface having a columnar structure in which a plurality of pillars are formed or a surface having a sieve structure or a network structure in which a plurality of pores are formed, but is not limited to these examples. .

In the apparatus of the present invention, the non-planar solid support may have any shape. For example, a solid support having a pillar structure having a plurality of pillars formed on a surface thereof, a solid support having a bead shape, and a sieve structure having a plurality of pores formed on a surface thereof. It may be selected from the group consisting of a solid support. The solid support may be used alone or as a collection of a plurality of the solid supports (eg, an aggregate packed in a tube or a container).

In the apparatus of the present invention, the solid support may be in the form of the inner wall of the microchannel or microchamber in the microfluidic device. Accordingly, the microbial separation device of the present invention may be a fluidic device or a microfluidic device having one or more inlets and outlets, the inlets and outlets communicating through channels or microchannels. have.

In the apparatus of the present invention, the solid support may have a columnar structure in which a plurality of pillars are formed on the surface thereof. The manufacture of pillars on solid supports is well known in the art. For example, fine pillars can be formed at high density using a photolithography process or the like used in a semiconductor manufacturing process. Preferably, the pillar has an aspect ratio of 1: 1 to 20: 1, but is not limited thereto. In the present invention, "aspect ratio" means the ratio of the cross-sectional diameter to the height of the column. The pillar structure may be a ratio of the distance between the pillar height and the pillar is 1: 1 to 25: 1. The pillar structure may have a distance between the pillars in a range of 5 μm to 100 μm.

In the apparatus of the present invention, the non-planar solid support may have a hydrophobicity with a water contact angle of 70 ° to 95 °. The hydrophobicity with a water contact angle of 70 ° to 95 ° consists of octadecyldimethyl (3-trimethoxysilyl propyl) ammonium (OTC) and tridecafluorotetrahydrooctyltrimethoxysilane (DFS). The compound selected from the group may be coated and given. The surface having a water contact angle of 70 ° to 95 ° preferably has octadecyldimethyl (3-trimethoxysilyl propyl) ammonium (OTC) and tridecafluorotetrahydrooctyltrimethoxy in the SiO ₂ layer of the solid support. Compounds selected from the group consisting of silanes (DFS) may be self-assembled monolayer (SAM) coated.

In the device of the present invention, the non-planar solid support may have one or more amine-based functional groups on its surface. Surface having at least one functional group of the amine-based may be a polyethyleneimine trimethoxysilane (PEIM) is coated on the surface of the solid support. The coated surface may preferably be a self-assembled monomolecular (SAM) coating of polyethyleneiminetrimethoxysilane (PEIM) on the SiO ₂ layer of the solid support.

In the apparatus of the present invention, the solid support may include a support of any material as long as it has water contact angle characteristics as described above or one or more amine-based functional groups on its surface. For example, glass, silicon wafers, plastic materials, and the like are included, but are not limited to these examples. The surface having a water contact angle of 70 ° to 95 ° or a surface having at least one amine-based functional group on the surface thereof is considered to be attached to the microorganism when the sample containing the microorganism contacts the solid support having the surface. The present invention is not limited to a specific mechanism.

In the apparatus of the present invention, the container in which the solid support is included may be any type of container. For example, it may be in the form of a chamber, channel, or column. One embodiment of the device of the present invention is a device in which the bead-shaped solid support is filled in a column.

The apparatus of the present invention also includes a microbial sample inlet and a buffer reservoir provided in the container. The buffer reservoir includes a buffer that can be used to adjust or dilute the microbial sample to pH 3.0 to 6.0. The buffer can be, for example, acetate buffer or phosphate buffer. The injection port and the reservoir are in fluid communication with the interior of the container.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

Example  1: Effect of Buffer Type and Concentration on Bacterial Concentration Using Solid Supports with Column Arrays in Flow Devices

In the present embodiment, the solids are flowed while the sample including the bacterial cells is flowed into a flow apparatus having an inlet and an outlet and a chamber in which an array of pillars is formed on a silicon chip having a size of 10 mm x 23 mm. The number of cells in the sample flowing out and adhered to the support was determined via colony counting, from which the bacterial cell capture efficiency by the solid support was calculated. In the pillar array, the distance between the pillars is 12 µm, the pillar height is 100 µm, and the cross section of each pillar has a square length of 25 µm on one side.

The surface of the region where the pillar array is formed has a surface coated with OTC (water contact angle 80 °) having a water contact angle in a range of 70 ° to 95 °.

The bacterial samples used were suspended in 1.0 × OD ₆₀₀ Escherichia coli samples in LB medium in 1 × PBS at pH 7.0 and diluted 100-fold with respective buffers to prepare 0.01 OD samples. The sample was adjusted to pH 4.0 using 100 mM sodium phosphate buffer (pH 4.0), 100 mM sodium acetate buffer (pH 4.0) and 100 mM sodium citrate buffer (pH 4.0), 200 μl flow rate 300 Flow from the inlet through the chamber to the outlet at μl / min. The experiment was repeated three times each.

1 is a diagram showing the effect of the attachment of cells to a solid support having a type of buffer present in the flow apparatus and having a column array formed thereon. As shown in FIG. 1, sodium phosphate buffer and sodium acetate buffer had high efficiency in attaching bacterial cells to the solid support, even among buffers having buffering power at low pH.

FIG. 2 shows the effect of salt concentration on cell attachment to a solid support having a surface in which a column array is formed and present in a flow apparatus. As shown in FIG. 2, the lower the salt concentration, the higher the efficiency of attaching the cells to the solid support. However, real samples, such as biological samples, require a buffer solution with a certain level of buffering to maintain a range of pH 3.0 to 6.0. Given this, salt concentrations suitable for attaching the cells to the solid support with high efficiency are 10 mM to 500 mM, preferably 50 mM to 300 mM (data not shown).

Example  2: Concentration of Bacterial Cells in Blood Using Solid Supports with Column Arrays in Flow Devices

In this embodiment, the sample containing bacteria cells is flowed into a flow apparatus having an inlet and an outlet and having a chamber having a surface having an array of pillars formed on a silicon chip having a size of 10 mm x 23 mm. Cells were attached onto the solid support and the number of bacterial cells in the outflowing sample was determined through colony counting, from which bacterial cell capture efficiency by the solid support was calculated. In the pillar array, the distance between the pillars is 12 µm, the pillar height is 100 µm, and the cross section of each pillar has a square length of 25 µm on one side.

The surface of the region where the pillar array is formed has a surface having a SiO ₂ layer and a surface coated with PEIM, OTC, and DFS, respectively, on the SiO ₂ layer.

10 μl of 1.0 OD Escherichia coli suspended in 1 × PBS (pH 7.0) was added to 990 μl of a mixture of 450 μl of blood and buffer (pH 3.0, pH 7.0), respectively, to prepare a 0.01 OD blood diluted sample.

The diluted blood sample flowed 200 μl from the inlet to the outlet through the chamber at a flow rate of 200 μl / min. Next, 100 mM sodium acetate (pH 4.0) was washed at a flow rate of 200 μl / min by flowing 300 μl from the inlet to the outlet through the chamber. The experiment was repeated three times each.

FIG. 3 is a diagram showing the effect of E. coli attachment to a solid support having pH and surface characteristics on which an array of pillars is formed and present in the flow apparatus and having a surface on which pillar arrays are formed. As shown in FIG. 3, when using a sample having a low pH, the efficiency of adhering to a solid support was high. On the other hand, the effect of the surface of the solid support surface on the cell adhesion was relatively weak. However, the electrostatic properties are weak and the surface tension is greater than the electrostatic properties SiO ₂ present or lower surface tension, that showed excellent characteristics in the same low pH is relatively hydrophobic surface.

FIG. 4 shows a sample of blood diluted with sodium acetate buffer (pH 5.2) flowing through a solid support having a surface present in the flow apparatus and having a columnar array formed therein, and flushed with 100 mM sodium acetate (pH 4.0) After that, the surface on which the pillar array is formed is observed by an optical electron microscope. E. coli cells were not eluted when quantified through colony counts. In addition, as shown in Figure 4, it can be visually confirmed that the animal cell, such as red blood cells that can act as a PCR inhibitor by the washing process was removed.

Example  3: Bacterial Cell Adhesion in Urine Using a Flat Solid Support

In this embodiment, a sample containing bacterial cells is placed on a silicon chip having a surface of a flat SiO ₂ layer having a size of 25.4 mm x 25.4 mm, and a substrate having a surface coated with PEIM, OTC, and DFS, respectively. Bacterial cells were added on the solid support, washed, and observed for attachment of bacterial cells through a microscope.

10 μl of 1.0 OD Escherichia coli suspended in 1 × PBS (pH 7.0) was added to 990 μl of a mixture of 495 μl of buffer (pH 3.0, pH 7.0) and urine, respectively, to prepare a 0.01 OD sample.

The diluted urine sample was placed on the patch-covered substrate and left for 5 minutes, and then each buffer, i.e., sodium acetate buffer, was used as sodium acetate buffer for 5 minutes, and sodium phosphate buffer was used for sodium phosphate buffer. Washed with.

5 is a diagram showing the effect on the solid support adhesion of E. coli in the sample according to the diluted urine sample pH. As shown in Figure 5, it can be seen that the adhesion of E. coli to the solid support is significantly affected by pH. In addition, at pH 6.0 or above, it can be seen that E. coli is not attached to the solid support. Therefore, in order to isolate bacterial cells, it is preferable to carry out at pH 6.0 or less.

Example  4: concentration of bacterial cells in urine using a solid support having a surface on which pillar arrays are formed

In this embodiment, the cells are introduced while flowing a sample containing bacterial cells into a flow apparatus having a chamber having a surface having an inlet and an outlet and having a surface having an array of pillars formed on a silicon substrate having a size of 10 mm x 23 mm. The number of cells in the sample flowing out attached to the solid support was determined via colony counting, from which the bacterial cell capture efficiency by the solid support was calculated. In the pillar array, the distance between the pillars is 12 µm, the pillar height is 100 µm, and the cross section of each pillar has a square length of 25 µm on one side.

The surface of the region where the pillar array is formed has a SiO ₂ layer and a surface coated with PEIM on the SiO ₂ layer, respectively.

10 μl of 1.0 OD Escherichia coli suspended in 1 × PBS (pH 7.0) was added to 990 μl of a mixture of 495 μl of buffer (pH 3.0, pH 7.0) and urine, respectively, to prepare a 0.01 OD sample.

The diluted urine sample flowed 200 μl from the inlet to the outlet through the chamber at a flow rate of 200 μl / min. Next, 100 mM sodium acetate (pH 4.0) was washed at a flow rate of 200 μl / min by flowing 300 μl from the inlet to the outlet through the chamber. The experiment was repeated three times each.

FIG. 6 shows the effect of pH and surface properties on the adhesion of E. coli in a diluted urine sample to a solid support having a surface on which a columnar array is formed. As shown in Figure 6, the adhesion efficiency of E. coli at pH 4.7 was high. The cell capture efficiency measured in this example is shown in Table 1 below.

Table 1.

Dilution buffer and final pH Acetate buffer (pH3) final pH 4.7 Phosphate Buffer (pH3) Final pH 6.1 Surface Characteristics (Water Contact Angle) SiO2 (<10 °) PEIM (about 35 °) SiO ₂ (<10 °) PEIM (about 35 °) Capture efficiency (%) 4 30 11 0

Example  5: Concentration of bacterial cells in urine using a solid support having a surface on which columnar arrays are formed: On variability  The concentration effect

In this embodiment, the cells are introduced while flowing a sample including bacterial cells into a flow apparatus having a chamber having an inlet and an outlet and having a surface having an array of pillars formed on a silicon chip having a size of 10 mm x 23 mm. The number of cells in the sample flowing out attached to the solid support was determined via colony counting, from which the cell capture efficiency by the solid support was calculated. In the pillar array, the distance between the pillars is 12 µm, the pillar height is 100 µm, and the cross section of each pillar has a square length of 25 µm on one side.

The surface of the region where the pillar array is formed has a surface coated with PEIM on the SiO ₂ layer.

10 μl of 1.0 OD Escherichia coli suspended in 1 × PBS (pH 7.0) was added to 990 μl of a mixture of 495 μl of buffer (pH 3.0, pH 7.0) and urine, respectively, to prepare a 0.01 OD sample.

The diluted urine sample flowed 200 μl from the inlet to the outlet through the chamber at a flow rate of 200 μl / min. Next, 100 mM sodium acetate (pH 4.0) was washed by flowing 300 μl from the inlet to the outlet through the chamber at a flow rate of 200 μl / min. The cell count counted the cells in the effluent flowing through the outlet before and after the urine sample was poured into the solid support.

Fig. 7 shows the result of concentrating E. coli cells in urine using a solid support having a surface on which columnar arrays are formed, showing the result of variation in urine. As shown in FIG. 7, the cell capture efficiency was significantly different depending on the type of urine. It is believed that the concentration of salt and other components depending on the state of the body and the type of ingested food cause the difference in cell capture efficiency, but the present invention is not limited to a specific mechanism. However, pH and conductivity do not seem to correlate with this difference in capture efficiency (data not shown).

Example  6: Concentration of bacterial cells in urine using a solid support having a surface on which pillar arrays are formed: Urinary  Determination of concentration effect by dilution and washing

In this embodiment, bacterial cells are introduced while flowing a sample containing bacterial cells into a flow apparatus having a chamber having a surface having an inlet and an outlet and having a surface having an array of pillars formed on a silicon chip having a size of 10 mm x 23 mm. The number of bacterial cells in the sample flowing out and adhered to the solid support was determined via colony counting, from which the bacterial cell capture efficiency by the solid support was calculated. In the pillar array, the distance between the pillars is 12 µm, the pillar height is 100 µm, and the cross section of each pillar has a square length of 25 µm on one side.

The surface of the region where the pillar array is formed has a surface coated with PEIM and OTC, respectively, on the SiO ₂ layer.

The cell sample was mixed 1: 1 with 100 mM sodium acetate buffer (pH 3.0) containing E. coli 0.01 OD ₆₀₀ , a sample having a final pH of 3.97 (hereinafter referred to as '1/2 dilution sample'), 0.01 OD 1: 4 with 100 mM sodium acetate buffer (pH 4.0) containing ₆₀₀ E. coli, containing a sample having a final pH of 4.05 (hereinafter referred to as '1/5 dilution sample'), and E. coli 0.01 OD ₆₀₀ Was mixed 1: 100 with 100 mM sodium acetate buffer (pH 4.0), and a sample having a final pH of 4.05 (hereinafter referred to as '1/7 dilution sample') was used.

The diluted urine sample flowed 200 μl from the inlet to the outlet through the chamber at a flow rate of 200 μl / min. Next, 100 mM sodium acetate (pH 4.0) was washed by flowing 300 μl from the inlet to the outlet through the chamber at a flow rate of 200 μl / min. The experiment was repeated three times each. The cell count was counted for the E. coli cells in the effluent flowing through the outlet before and after the urine sample flowed to the solid support.

FIG. 8 shows the result of concentration of cells in urine using a solid support having a surface on which pillar arrays are formed, showing the result of dilution of urine. As shown in FIG. 8, as the dilution fold increased, cell capture efficiency greatly increased. There was little cell elution in the washing process.

Example  7: Concentration of Escherichia Coli in Urine Simulation Solution Using Solid Support with Surface with Pillar Array Formation: Screening for Factors Interrupting Cell Capture

In this embodiment, the cells are introduced while flowing a sample including bacterial cells into a flow apparatus having a chamber having an inlet and an outlet and having a surface having an array of pillars formed on a silicon chip having a size of 10 mm x 23 mm. The number of bacterial cells in the sample flowing out and adhered to the solid support was determined via colony counting, from which the bacterial cell capture efficiency by the solid support was calculated. In the pillar array, the distance between the pillars is 12 µm, the pillar height is 100 µm, and the cross section of each pillar has a square length of 25 µm on one side.

The surface of the region where the pillar array is formed has an OTC SAM coated surface on a SiO ₂ layer.

The cell sample used a solution based on sodium acetate buffer and a solution based on dialysis urine, including E. coli 0.01 OD ₆₀₀ .

The solution (pH 4.0) based on the sodium acetate buffer is as follows:

Buffer: 100 mM sodium acetate buffer (pH 4.0).

Buffer + salt: 100 mM sodium acetate buffer so that the (pH 4.0) to the salt concentration, 88 mM NaCl, 67 mM KCl , 38 mM NH 4 Cl and _{18 mM Na 2 SO 4, NaCl} 0.514 g, KCl 0.5 g, NH 4 A solution obtained by adding 0.203 g of Cl and 0.259 g of Na ₂ SO ₄ .

Buffer + Salt + Urea: A solution comprising 333 mM urea in the "Buffer + Salt" solution.

Buffer + Salt + Urea + Creatine: A solution comprising 333 mM urea and 9.8 mM creatine in the "Buffer + Salt" solution.

Buffer + Salt + Urea + Creatine + Uric Acid: A solution comprising 333 mM urea and 9.8 mM creatine and 2.5 mM uric acid in the "buffer + salt" solution.

Buffer + Salt + Urea + Creatine + Uric Acid + Glucose: A solution comprising 333 mM urea and 9.8 mM creatine and 2.5 mM uric acid and 0.6 mM glucose in the "buffer + salt" solution.

The dialysis-based solution was obtained by mixing 1: 1 of dialysis urine with each solution based on 2 × the sodium acetate buffer, with a final pH of 3.97.

The urine sample flowed 200 μl from the inlet to the outlet through the chamber at a flow rate of 200 μl / min. The experiment was repeated three times each. The cell count counted the cells in the effluent flowing through the outlet before and after the urine sample was flowed to the solid support.

Fig. 9 shows the result of concentration of cells in urine using a solid support having a surface on which pillar arrays are formed. It can be seen that the capture efficiency is lowered by the concentration of salts present in the urine and by other substances (mainly creatine). This can be sufficiently improved by the salt removal device (electrodialysis and zeolite treatment).

Example  8: Concentration of E. coli in urine using a solid support having a surface on which columnar arrays are formed: Urinary  Determination of concentration effect according to dilution rate and flow rate

In this embodiment, bacterial cells are introduced while flowing a sample containing bacterial cells into a flow apparatus having a chamber having a surface having an inlet and an outlet and having a surface having an array of pillars formed on a silicon chip having a size of 10 mm x 23 mm. The number of cells in the sample flowing out on the solid support was determined via colony counting, from which bacterial cell capture efficiency by the solid support was calculated. In the pillar array, the distance between the pillars is 12 µm, the pillar height is 100 µm, and the cross section of each pillar has a square length of 25 µm on one side.

The surface of the region where the pillar array is formed has a surface coated with PEIM on the SiO ₂ layer.

Samples were prepared as follows. The buffer and urine were mixed to a final volume of 1 ml while adjusting the dilution scale. 10 μl of 1.0 OD Escherichia coli was added thereto.

The diluted urine sample flowed 200 μl from the inlet to the outlet through the chamber at a flow rate of 200 μl / min. The experiment was repeated three times each. Bacterial cell counts counted the cells in the effluent flowing through the outlet before and after the urine sample was poured into the solid support.

FIG. 10 shows the results of concentration of cells in urine using a solid support having a surface on which pillar arrays are formed, showing the results of dilution and flow rate of urine. As shown in FIG. 10, as the dilution fold increased, the cell capture efficiency increased, and as the flow rate increased, the cell capture efficiency decreased.

According to the method for separating the microorganism of the present invention, microbial cells including bacterial cells, fungal cells or viruses present in a biological sample can be efficiently separated.

According to the device for isolating the microorganism of the present invention, it is possible to efficiently separate microbial cells including bacterial cells, fungal cells or viruses.

Claims (26)

A method for separating microorganisms comprising contacting a non-planar solid support with a sample containing microorganisms in a range of pH 3.0 to 6.0, wherein the non-planar solid support has pillars formed on a surface thereof. A solid support having a columnar structure, wherein the pillar has an aspect ratio of 1: 1 to 20: 1, the ratio of the height of the pillar to the distance of the pillar is 1: 1 to 25: 1, and the distance between the pillars. Is selected from the group consisting of 5 μm to 100 μm. The method of claim 1, wherein the microorganism is a bacterium, fungus or virus. The method of claim 1, wherein the sample is a biological sample. The method of claim 3, wherein the sample is blood, urine, or saliva. The method of claim 1, wherein the sample is diluted with phosphate buffer or acetate buffer. The method of claim 5, wherein the dilution is performed at a ratio of 1: 1 to 1:10. The method of claim 5, wherein the sample has a salt concentration of 10 mM to 500 mM. 8. The method of claim 7, wherein the sample has a salt concentration of 50 mM to 300 mM. delete delete delete delete delete A method for separating microorganisms comprising contacting a non-planar solid support with a sample containing microorganisms in a range of pH 3.0 to 6.0, wherein the non-planar solid support has a water contact angle of 70 ° to 95 °. Phosphorus hydrophobic or having an amine-based functional group on its surface, wherein the microorganism is a bacterium, fungus or virus is a method for separating the microorganism, wherein the hydrophobicity is the octadecyldimethyl (3- A process characterized by coating with a compound of trimethoxysilyl propyl) ammonium (OTC) or tridecafluorotetrahydrooctyltrimethoxysilane (DFS). delete A method for separating microorganisms comprising contacting a non-planar solid support with a sample containing microorganisms in the range of pH 3.0 to 6.0, wherein the non-planar solid support is characterized by Wherein the microorganism is a bacterium, fungus or virus, wherein the surface having the amine-based functional group is coated with polyethyleneiminetrimethoxysilane (PEIM). The method of claim 1 further comprising washing material other than the desired microorganism that is not attached to the solid support after the contacting step. A microorganism separation device comprising a container including a non-planar solid support, a sample injection unit provided in the container, and a buffer solution storage unit provided in the container, wherein the non-planar solid support has a pillar on the surface thereof. ) Is a solid support having a columnar structure, the pillar has an aspect ratio of 1: 1 to 20: 1, the ratio of the distance between the pillar height and the pillar is 1: 1 to 25: 1 The microbial separation device is selected from the group consisting of having a distance between 5 μm and 100 μm. delete delete delete delete A microorganism separation device comprising a container including a non-planar solid support, a sample injection unit provided in the container, and a buffer solution storage unit provided in the container, wherein the non-planar solid support has a water contact angle of 70 °. Device having a hydrophobicity of 95 to 95 degrees or having an amine-based functional group on the surface, the microorganism is a bacteria, fungus or virus. The method of claim 23, wherein the hydrophobicity is coated on the surface of the solid support with a compound of octadecyldimethyl (3-trimethoxysilyl propyl) ammonium (OTC) or tridecafluorotetrahydrooctyltrimethoxysilane (DFS). Apparatus characterized in that the property is given by. delete The device of claim 23, wherein the surface having amine-based functional groups is coated with polyethyleneiminetrimethoxysilane (PEIM).

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