US20080044884A1

US20080044884A1 - Method and device for separating cells from a sample using a nonplanar solid substrate

Info

Publication number: US20080044884A1
Application number: US11/841,089
Authority: US
Inventors: Kyu-youn Hwang; Sung-young Jeong; Joon-Ho Kim; Hun-joo Lee; Jung-Im Han
Original assignee: Samsung Electronics Co Ltd
Current assignee: Samsung Electronics Co Ltd
Priority date: 2006-08-21
Filing date: 2007-08-20
Publication date: 2008-02-21
Also published as: EP1892295B1; US20080044864A1; US20080070282A1; EP1892295A2; JP2008048735A; US7919278B2; EP1900807A2; EP1900807A3; EP1972688A1; EP1900807B1; EP1972688B1; DE602007006908D1; EP1892295A3

Abstract

Provided is a method and device for separating a cell from a sample. The method comprises contacting a nonplanar solid substrate with a cell-containing sample in a liquid medium having a pH of 3.0 to 6.0.

Description

This application claims priority to Korean Patent Application Nos. 10-2006-0079053, 10-2006-0079054, 10-2006-0079055, and 10-2006-0079056, each filed on Aug. 21, 2006, the disclosure of each is incorporated herein in its entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention relates to a method and device for separating a microorganism using a nonplanar solid substrate.
2. Description of the Related Art
Conventionally, a microorganism is separated from a sample by centrifugation and filtering. In addition, to concentrate or separate a specific cell, the cell can be specifically bound to a substrate to which a receptor or ligand that specifically binds to the specific cell is bound. For example, a sample that contains the specific cell is contacted with a substrate, to which an antibody that binds to a specific protein of the cell is bound, so that the cell binds to the antibody and unbound cells can be washed off the substrate. Such a method is an affinity chromatography technique.
Furthermore, Korean Laid-open Patent Publication No. 2006-0068979 discloses a device for separating a cell using an ultrasound field and traveling wave dielectrophoresis. The device includes: a piezoelectric transducer, which is connected to the ends of an upper glass substrate, and which converts an external electric input into a mechanical vibration for application to the upper glass substrate; and a cell separation device in which a number of electrodes (“n”) are disposed on a lower substrate parallel to the upper glass substrate, each electrode is perpendicular to the lengthwise direction of the piezoelectric transducer, and the “n” number of electrodes are disposed at constant intervals in the lengthwise direction of the piezoelectric transducer. A fluid comprising a cell can fill the space between the upper glass substrate and the lower substrate.
However, these conventional techniques are based on fixing a ligand or a receptor to a solid substrate or on use of an external operation power to selectively concentrate or separate a specific cell. Accordingly, a method or device for separating a cell using the characteristics of a solid substrate itself and conditions of a liquid medium is not known.

SUMMARY OF THE INVENTION

The present invention provides a method of separating a cell from a sample comprising contacting a nonplanar solid substrate with a sample, wherein the sample comprises a cell and a liquid medium having a pH of 3.0 to 6.0, wherein the contacting is such that cells in the sample are separated from the sample.
The present invention also provides a device for separating a cell from a sample comprising a container comprising a nonplanar solid substrate; a sample inlet; and a buffer solution storage unit.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features and advantages of the present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which:

FIG. 1 is a graph illustrating effects of buffer type on binding of E. coli onto a solid substrate having a surface with a pillar array of a fluidic device;

FIG. 2 is a graph illustrating effects of concentration of buffer, at constant pH, on binding of E. coli onto a solid substrate having a surface with a pillar array of a fluidic device;

FIG. 3 is a graph illustrating effects of buffer pH and surface characteristics of the pillar array on binding of E. coli contained in blood onto a solid substrate having a surface with a pillar array of a fluidic device;

FIG. 4 presents optical microscopic images of a surface with a pillar array of a solid substrate of a fluidic device after a blood sample (pH 5.2) diluted with an acetate buffer was contacted with the solid substrate; images before and after washing with 100 mM sodium acetate (pH 4.0) are shown;

FIG. 5 presents optical microscopic images showing the effects of pH and surface characteristics of the pillar array on binding of E. coli contained in a diluted urine sample onto a solid substrate;

FIG. 6 is a graph illustrating effects of pH and surface characteristics of the pillar array on binding of E. coli contained in a urine sample diluted with a buffer onto a solid substrate having a surface with a pillar array;

FIG. 7 is a graph illustrating results of binding of E. coli contained in various urine samples onto a solid substrate having a surface with a pillar array of a fluidic device;

FIG. 8 is a graph illustrating effects of urine dilution on binding of E. coli contained in diluted urine samples onto a solid substrate having a surface with a pillar array;

FIG. 9 is a graph illustrating effect of components of the E. coli-containing sample on binding of E. coli from the sample onto a solid substrate having a surface with a pillar array; and

FIG. 10 is a graph illustrating the effect of urine dilution ratio and flow rate on binding of E. coli contained in a urine sample onto a solid substrate having a surface with a pillar array.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown.
A method of separating a cell from a sample according to an embodiment of the present invention comprises contacting a cell-containing sample with a nonplanar solid substrate in a liquid medium having a pH of 3.0 to 6.0.
The method according to an embodiment of the present invention includes contacting a cell-containing sample with a nonplanar solid substrate in a liquid medium having a pH of 3.0 to 6.0. By the contacting, the cell is bound to the solid substrate.
As used herein, the term “cell” means a micro-organism, a prokaryotic or eukaryotic cell (e.g., a plant cell, a bacteria cell, a pathogenic cell, or a yeast cell), an aggregate of cells, a virus, a fungus, or other nucleic acid containing biological material, such as, for example, an organelle.
As used herein, the term “nucleic acid” means DNA or RNA, or a combination of both. The DNA or RNA can be in any possible configuration, i.e., in the form of double-stranded (ds) nucleic acid, or in the form of single-stranded (ss) nucleic acid, or as a combination thereof (in part ds or ss).
When a cell is present in a liquid medium in contact with a solid substrate, the cell, such as bacteria, can exist in the liquid medium or can be bound to the solid substrate. The location of the cell may be determined by the difference in surface tension of the liquid medium, the solid substrate, and the cell. For example, when the liquid medium has a greater surface tension than the cell, the cell may be easily bound to a solid substrate having a surface tension smaller than the cell, for example, to a hydrophobic solid substrate. Similarly, when the liquid medium has a smaller surface tension than the cell, the cell may be easily bound to a solid substrate having a high surface tension, for example, to a hydrophilic solid substrate; and when the liquid medium and cell have the same surface tension, the surface tension does not affect the binding of the cell to the solid substrate and other interaction factors, such as electrostatic interactions, may affect such binding (see Applied and Environmental Microbiology, July 1983, p.90-97). In addition, it is known that a cell can be bound to a solid substrate based on electrostatic attractions as well as being thermodynamically driven by differences in surface tension. However, binding base on electrostatic attraction occur very slowly and the bound quantity is very small.
Inventors of the present invention made efforts to solve these problems of the conventional techniques and found that a cell could be separated from a sample in high yield by contacting a nonplanar solid substrate with the cell-containing sample having a pH in the range of pH 3.0 to 6.0. Without being limited by theory or mechanism, such a result may be because the nonplanar structure of the solid substrate increases the surface area of the solid substrate, and because by using a liquid medium with a pH of 3.0 to 6.0, the cell membrane of a cell is denatured and therefore the cell has a smaller solubility in the solution and relatively more cells are bound to the solid substrate surface.
During the contacting process, the sample can be any sample containing a cell. For example, the sample can be a biological sample containing a cell, a clinical sample containing a cell, or a lab sample containing a cell. In the present specification, the biological sample refers to a sample that comprises a cell or tissue, such as a cell, biological liquid, or tissue sample obtained from an individual. The individual can be an animal, specifically the individual can be a human. The biological sample can be saliva, sputum, blood, blood cell (for example, red blood cell or white blood cell), amniotic fluid, serum, semen, bone marrow, tissue or micro needle biopsy sample, urine, peritoneum fluid, pleura fluid, or cell cultures. In addition, the biological sample can be a tissue section, such as a frozen section taken for a histological object. Preferably, the biological sample is a clinical sample derived from a human patient. More preferably, the biological sample is blood, urine, saliva, or sputum.
In some embodiments, a cell that is to be separated is a bacterial cell, a fungus, or a virus.
During the contacting process, the biological sample can be diluted with a solution that may buffer the cell at a low pH or a buffer. The buffer can be a phosphate buffer, such as sodium phosphate of pH 3.0 to 6.0, or an acetate buffer, such as sodium acetate of pH 3.0 to 6.0. The degree of dilution of the biological sample is not limited. In some embodiments, the biological sample is diluted in a range of 1:1 to 1:1,000, and preferably, in a range of 1:1 to 1:10.
During the contacting process, the sample may have a salt concentration of 10 mM to 500 mM, and preferably, 50 mM to 300 mM. That is, the sample may have an acetate or phosphate ion concentration of 10 mM to 500 mM, preferably 50 mM to 300 mM.
During the contacting process, the solid substrate has a nonplanar shape so that the surface area of the solid substrate is increased compared to a plane. For example, the solid substrate may have a corrugated surface. Herein, a “corrugated surface” refers to an unlevel surface having grooves and ridges. The corrugated surface can be a surface with a plurality of pillars or a sieve-shaped surface with a plurality of pores. However, the corrugated surface may have other shapes.
During the contacting process, the nonplanar solid substrate may have various shapes. For example, the nonplanar solid substrate can be selected from a solid substrate having a surface with a plurality of pillars, a bead-shaped solid substrate, and a sieve-shaped solid substrate having a plurality of pores at its surface. The solid substrate can be a single solid substrate or a combination of solid substrates, such as a solid substrate assembly which fills a tube or container.
During the contacting process, the solid substrate may form an inner wall of a microchannel or microchamber of a microfluidic device. Accordingly, the method according to an embodiment of the present invention can be used in a fluidic device or microfluidic device having at least one inlet and outlet connected through a channel or microchannel.
As used herein, the term “microfluidic device” incorporates the concept of a microfluidic device that comprises microfluidic elements such as, e.g., microfluidic channels (also called microchannels or microscale channels). As used herein, the term “microfluidic” refers to a device component, e.g., chamber, channel, reservoir, or the like, that includes at lest one cross-sectional dimension, such as depth, width, length, diameter, etc. of from about 0.1 micrometer to about 1000 micrometer. Thus, the term “microchamber” and “microchannel” refer to a channel and a chamber that includes at lest one cross-sectional dimension, such as depth, width, and diameter of from about 0.1 micrometer to about 1000 micrometer, respectively.
In an embodiment of the method according to the present invention, during the contacting process, the solid substrate has a surface having a plurality of pillars. Methods of forming pillars on a solid substrate are well known in the art. For example, micro-pillars can be formed at a high density using a photolithography process used in a semiconductor manufacturing process. The micro-pillars may have an aspect ratio of 1:1 to 20:1. However, the aspect ratio of the micro-pillars is not limited thereto. In the present specification, the aspect ratio refers to the ratio of the cross-sectional diameter to the height of a pillar. In the pillar structure, the ratio of the pillar height to the distance between adjacent pillars may be in the range of 1:1 to 25:1. The distance between adjacent pillars may be in the range of 5 μm to 100 μm.
In the method, during the contacting process, the nonplanar solid substrate may have a water contact angle of 70° to 95° and thus be hydrophobic. The hydrophobic property of the solid substrate having a water contact angle of 70° to 95° can be obtained by coating the solid substrate with octadecyidimethyl(3-trimethoxysilyl propyl)ammonium (OTC) or tridecafluorotetrahydrooctyltrimethoxysilane (DFS). More specifically, the surface having a water contact angle of 70° to 95° can be obtained by self-assembled molecule (SAM) coating octadecyidimethyl(3-trimethoxysilyl propyl)ammonium (OTC) or tridecafluorotetrahydrooctyltrimethoxysilane (DFS) on a SiO₂layer of the solid substrate.
In the method, during the contacting process, the nonplanar solid substrate may have at least one amine-based functional group at its surface. The surface with the amine-based functional group may be obtained by coating polyethyleneiminetrimethoxysilane (PEIM) on the solid substrate. For example, the coated surface can be obtained by coating polyethyleneiminetrimethoxysilane (PEIM) on a SiO₂layer of the solid substrate using a self-assembled molecule (SAM) coating process.
In this application, the term “water contact angle” refers to water contact angle measured by a Kruss Drop Shape Analysis System type DSA 10 Mk2. A droplet of 1.5 μl deionized water is automatically placed on the sample. The droplet was monitored every 0.2 seconds for a period of 10 seconds by a CCD-camera and analyzed by Drop Shape Analysis software (DSA version 1.7, Kruss). The complete profile of the droplet was fifted by the tangent method to a general conic section equation. The angles were determined both at the right and left side. An average value is calculated for each drop and a total of five drops per sample are measured. The average of the five drops is taken the contact angle.
In the method, during the contacting process, the solid substrate can be a substrate formed of any kind of material that has the water contact angle described above or has at least one amine-based functional group at its surface, or of any kind of material which has a surface which may be coated as described above to obtain the water contact angel described above or to have at least one amine-based functional group at its surface. For example, the solid substrate can be formed of glass, silicon wafer, plastic, or the like, but is not limited thereto.
When a solid substrate having a surface having the water contact angle of 70° to 95° or a surface having at least one amine-based functional group is contacted with a sample containing a cell, the cell is assumed to bind to the solid substrate. However, the present invention is not limited to such a specific mechanism.
The method may further comprise, after the contacting process, washing away other materials in the sample, excluding the target cell, which are not bound to the solid substrate with a wash buffer. During the washing process, any solution that does not remove the bound target cell from the solid substrate but does remove unbound impurities which may adversely affect subsequent processes can be used. For example, an acetate buffer or a phosphate buffer which is used as a binding buffer can be used as the washing solution. The washing solution may have a pH of 3.0 to 6.0.
In the present specification, “separation of a cell” intends to include concentrating the cell in the sample or isolating the cell from the sample.
The concentrated cells bound to the solid substrate according to an embodiment of the present invention can be used without any separation of the cells from the solid substrate in an additional process, such as cell elution or DNA isolation. Alternatively, the concentrated cells bound to the solid substrate can be eluted from the solid substrate. Either cells bound to the solid substrate or eluted cells can be used in a subsequent process.
Accordingly, an embodiment of the method according to the present invention may further comprise eluting the bound cell from the solid substrate, after the contacting step and/or the washing step. In the eluting process, an eluting solution used can be any solution known in the art that frees the microorganism from the solid substrate. For example, the eluting solution is water or tris buffer. The pH of the eluting solution can be 6.0 or higher.
A device for separating a cell from a sample according to an embodiment of the present invention comprises a container comprising a nonplanar solid substrate; a sample inlet; and a buffer solution storage unit.
The characteristics of a solid substrate suitable for use in the device are those described above in the discussion of the method.
In the device, the container comprising the solid substrate may have various shapes, and can be, for example, a chamber, a channel, or a column. The device may comprise a column filled with bead-shaped solid substrates.
In the device, the sample inlet and the buffer storage unit are in fluid communication with the inside of the container.
The present invention will be described in further detail with reference to the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

EXAMPLES

Example 1

Effects of Kind and Concentration of Buffer on Capturing Bacteria using a Solid Substrate having a Pillar Array in a Fluidic Device

In the current example, a bacteria-containing sample was loaded to a fluidic device including an inlet, an outlet, and a chamber comprising a 10 mm×23 mm silicon chip with a pillar array so that the bacterial cells were bound to the solid substrate. The number of cells in the bacteria-containing sample before and after elution through the fluidic device was determined through colony counting in order to calculate the bacteria capturing efficiency of the solid substrate. In the pillar array, the distance between adjacent pillars was 12 μm, the height of each pillar was 100 μm, and the sectional surface of each pillar was a regular square with each side of 25 μm.
Also, the pillar array had a surface coated with OTC having a water contact angle of 80°. Coatings of OTC having a water contact angle of 70° to 95° provide similar effects on cell binding and separation from the sample as those described below for an OTC coating with a water contact angle of 80°.
The bacteria sample used was prepared by suspending an E. coli sample in LB medium in 1× phosphate buffered saline (PBS) at pH 7.0 to achieve of OD₆₀₀=1.0. The suspended sample was then diluted 100-fold with a desired buffer to obtain a sample with 0.01 OD₆₀₀. The sample was adjusted to have a pH of 4.0 by diluting with 100 mM sodium phosphate buffer (pH 4.0), 100 mM sodium acetate buffer (pH 4.0), or 100 mM sodium citrate buffer (pH 4.0). 200 μl of each of the adjusted samples was loaded into the fluidic device at a flow rate of 300 μl/minute. This experiment with each buffer was repeated three times.
FIG. 1 is a graph illustrating the effect of the buffer type on binding of E. coli onto the solid substrate having a surface with a pillar array of the fluidic device. As illustrated in FIG. 1, among the three buffers having a buffering power in low pH, the sodium phosphate buffer and the sodium acetate buffer showed excellent capturing efficiency of the bacterial cell onto the solid substrate.
FIG. 2 is a graph illustrating effects of concentration of the buffer on binding of E. coli onto the solid substrate having a surface with a pillar array of the fluidic device. As illustrated in FIG. 2, when the concentration of the buffer was lower, the capturing efficiency of the bacterial cell onto the solid substrate was higher. However, a real sample, such as a biological sample, requires a buffer solution having adequate buffering power to maintain the pH of the sample in the range of 3.0 to 6.0. In consideration of such characteristics of a real sample, the buffer concentration required to obtain high capturing efficiency of the cells to the solid substrate may be in the range of 10 mM to 500 mM, more preferably, 50 mM to 300 mM (data not shown).

Example 2

Capturing Bacterial Cells in Blood using a Solid Substrate having a Pillar Array in a Fluidic Device

In the current example, a bacteria-containing sample was loaded to a fluidic device as described in Example 1. The number of cells in the bacteria-containing sample before and after elution through the fluidic device was determined through colony counting in order to calculate the bacteria capturing efficiency of the solid substrate.
Four different pillar arrays were prepared and tested in this experiment: a pillar array having a SiO₂layer at its surface, a pillar array having a SiO₂layer coated with PEIM at its surface, a pillar array having a SiO₂layer coated with OTC at its surface, and a pillar array having a SiO₂layer coated with DFS at its surface.
10 μl of 1.0 OD₆₀₀ E. coli suspended in 1×PBS (pH 7.0) was added to 990 μl of a solution which consisted of 495 μl of blood and 495 μl of buffer (pH 4.0 or pH 7.0) to prepare a diluted blood sample of 0.01 OD₆₀₀.
200 μl of the diluted blood sample was loaded into the fluidic device at a flow rate of 200 μl/minute. Then, 300 μl of 100 mM sodium acetate (pH 4.0) was loaded into the fluidic device at a flow rate of 200 μl/minute to wash unbound materials from the sample out of the fluidic device. This experiment was repeated three times.
FIG. 3 is a graph illustrating effects of pH and surface characteristics of the pillar array on binding of E. coli contained in blood onto a solid substrate having a surface with a pillar array. As illustrated in FIG. 3, a sample having low pH showed higher capturing efficiency onto the solid substrate than did a sample having high pH, regardless of the surface characteristics of the pillar array of the solid substrate. On the other hand, the effect of the surface characteristics of the pillar array of the solid substrate was relatively small. However, when compared at low pH, a relatively hydrophobic surface having relatively high electrostatic properties (PEIM) or low surface tension (OTC) showed higher capturing efficiency than the SiO₂surface having low electrostatic properties and high surface tension.
FIG. 4 are optical microscopic images of a surface of a solid substrate with a pillar array of a fluidic device, after a blood sample (pH 5.2) diluted with acetate buffer was contacted with the solid substrate and then washed with 100 mM sodium acetate (pH 4.0). Colony counting of the diluted blood sample eluted from the fluidic device indicated that no E. coli cells eluted from the solid substrate. In addition, as illustrated in FIG. 4, removal of an animal cell, such as a red blood cell, which can act as a PCR inhibitor during the washing process was able to be identified with a microscopy. In FIG. 4A, impurity materials shown between retangular structures is considered to animal cell such as red blood cell and white blood cell, and/or proteins. FIG. 4B. shows such materials except bacteria can be removed by a washing process. The method of present invention is directed to capture target bacterial cell after removing the materials. FIG. 4. shows that the removal of animal cells such as red blood cell and/or white blood cell, but not bacterial cell, can be identified with a microscopy.

Example 3

Binding of Bacterial Cells contained in Urine to a Planar Solid Substrate

In the current example, 25.4 mm×25.4 mm silicon chips comprising a SiO₂layer, a SiO₂layer coated with PEIM, a SiO₂layer coated with OTC, or a SiO₂layer coated with DFS, respectively, were contacted with a sample including a bacterial cell so that the bacterial cell was bound to the solid substrate and washed. The binding of the bacterial cell was identified using a microscope.
10 μl of 1.0 OD₆₀₀ E. coli suspended in 1×PBS (pH 7.0) was added to 990 μl of a solution which consisted of 495 μl of urine and 495 μl of buffer (pH 3.0 or pH 7.0) to prepare a diluted urine sample of 0.01 OD₆₀₀sample.
The diluted urine sample was added to the substrate, covered with a lid in the form of patch, and then left to sit for 5 minutes. Then, the liquid media was removed from the substrate and a washing solution was added to the substrate. The substrate was incubated for 5 min in the washing solution for washing the planar solid substrate. When the sodium acetate buffer was used, the sample was washed using the sodium acetate buffer for 5 minutes; and when the sodium phosphate buffer was used, the sample was washed using the sodium phosphate buffer.
FIG. 5 shows effects of pH of the diluted urine sample on binding of E. coli contained in the sample onto a solid substrate. As illustrated in FIG. 5, pH of the diluted urine sample significantly affected the binding of E. coli onto the solid substrate. In addition, it was found that E. coli was not bound to the solid substrate when the pH is 6.0 or higher. Accordingly, it is desired to perform separating of the bacterial cell in the pH of 6.0 or lower. Specifically, as shown in FIG. 5, the number of E. coli cells attached to the solid substate having a SiO₂layer, a SiO₂layer coated with PEIM, a SiO₂layer coated with OTC, or a SiO₂layer coated with DFS was about 97 cells, 95 cells, 56 cells, and 46 cells, respectively per 25.4 mm×25.4 mm (24 cells) silicon chips at pH 5.6 (upper panel), however, no E. coli cells were remain attached to the same solid substrate at pH 6.4 (lower panel). The number of E. coli cells attached to the solid substrate was determined by counting on the microscopy.

Example 4

Capturing Bacterial Cells in Urine using a Solid Substrate having a Surface with a Pillar Array

In the current example, a bacteria-containing sample was loaded to a fluidic device as described in Example 1 so that the bacterial cell was bound to the solid substrate. The number of cells in the bacteria-containing sample before and after elution through the fluidic device was determined through colony counting in order to calculate the bacteria capturing efficiency of the solid substrate. The dimensions of the pillar array were as described in Example 1.
A pillar array having a SiO₂layer at its surface and a pillar array having a SiO₂layer coated with PEIM at its surface were prepared.
10 μl of 1.0 OD₆₀₀ E. coli suspended in 1×PBS (pH 7.0) was added to 990 μl of a solution which consisted of 495 μl of urine and 495 μl of buffer (pH 3.0 or pH 7.0) to prepare a diluted urine sample of 0.01 OD₆₀₀sample.
200 μl of the diluted urine sample was loaded into the fluidic device at a flow rate of 200 μl/minute. Then, 300 μl of 100 mM sodium acetate (pH 4.0) was pumped through the fluidic device at a flow rate of 200 μl/minute to wash any unbound sample from the solid substrate. This experiment was repeated three times.
FIG. 6 is a graph illustrating the effects of pH and surface characteristics of the pillar array on binding of E. coli contained in the diluted urine sample onto the solid substrate having a surface with a pillar array. As illustrated in FIG. 6, when the pH was 4.7, the capturing efficiency of E. coli was highest for the pillar array coated with PEIM. The cell capturing efficiency measured according to the current example is shown in Table 1:

	TABLE 1

	Diluted Buffer and Final pH

	Acetate Buffer (pH3)	Phosphate Buffer (pH3)
	Final pH 4.7	Final pH 6.1

Surface	SiO₂	PEIM	SiO₂	PEIM
Characteristics
(Water Contact	(<10°)	(Approximately	(<10°)	(Approximately
Angle)		35°)		35°)
Capturing	4	30	11	0
Efficiency (%)

Example 5

Capturing Bacterial Cells in Urine using a Solid Substrate having a Surface with a Pillar Array: Concentration Effects with respect to Urine Variation

In the current example, a bacteria-containing sample was loaded to a fluidic device as described in Example 1 so that the bacterial cells bound to the solid substrate. The number of cells in the bacteria-containing sample before and after elution through the fluidic device was determined through colony counting in order to calculate the bacteria capturing efficiency of the solid substrate.
The pillar array had dimensions as described in Example 1 and had a SiO₂layer coated with PEIM at its surface.
10 μl of 1.0 OD₆₀₀ E. coli suspended in 1×PBS (pH 7.0) was added to 990 μl of a solution which consisted of 495 μl of urine and 495 μl of buffer (pH 3.0, pH 7.0) to prepare a diluted urine sample of 0.01 OD₆₀₀sample.
200 μl of the diluted urine sample was pumped through the fluidic device at a flow rate of 200 μl/minute. Then, 300 μl of 100 mM sodium acetate (pH 4.0) was pumped through the fluidic device at a flow rate of 200 μl/minute to wash unbound sample from the solid substrate. Capture efficiency was determined by counting the number of cells contained in the urine sample before the cell was contacted with the solid substrate and counting the number of cells in the urine sample eluting from the outlet.
FIG. 7 is a graph illustrating results of separating cells from the urine sample using the solid substrate having a surface with a pillar array of the fluidic device as a function of urine variation. As illustrated in FIG. 7, the cell capturing efficiency was significantly changed according to the kind of urine. Without being limited to any specific mechanism, such results are assumed to result from variation in the concentration of salt and other components in the urine according to body conditions and foods consumed. In addition, pH and conductivity of the urine are not directly related to change in capturing efficiency (data not shown).

Example 6

Capturing Bacterial Cells in Urine using a Solid Substrate having a Surface with a Pillar Array: Concentration Effects with respect to Dilution and Washing of Urine

In the current example, a bacteria-containing sample was loaded to a fluidic device as described in Example 1 so that the bacterial cell was bound to the solid substrate. The number of cells in the bacteria-containing sample before and after elution through the fluidic device was determined through colony counting in order to calculate the bacteria capturing efficiency of the solid substrate.
The pillar array used had dimensions as described in Example 1. A pillar array having a SiO₂layer coated with PEIM at its surface and a pillar array having a SiO₂layer coated with OTC were prepared.
The following cell samples were prepared: a sample with a final of pH 3.97 prepared by mixing urine with a 100 mM sodium acetate buffer (pH 3.0) solution, containing E. coli at 0.01 OD₆₀₀, at a 1:1 ratio (hereinafter, referred to as 1/2 diluted sample), a sample with a final of pH 4.05 prepared by mixing urine with a 100 mM sodium acetate buffer (pH 4.0) solution, containing E .coli of 0.01 OD₆₀₀, at a 1:4 ratio (hereinafter, referred to as 1/5 diluted sample, and a sample with a final of pH 4.05 prepared by mixing urine with—a 100 mM sodium acetate buffer (pH 4.0) solution, containing E. coli of 0.01 OD₆₀₀, at a 1:6 ratio (hereinafter, referred to as 1/7 diluted sample).
200 μl of each of these diluted urine samples was pumped through the fluidic device at a flow rate of 200 μl/minute. Then, 300 μl of 100 mM sodium acetate (pH 4.0) was pumped through the fluidic device at a flow rate of 200 μl/minute, for washing. This experiment was performed three times for each diluted urine sample. The capturing efficiency was determined by using colony counting to count the number of cells contained in the diluted urine sample before the cell was pumped through the fluidic device and contacted with the solid substrate and to count the number of cells in the diluted urine sample which emerged from the outlet after being pumped through the fluidic device. Cells were not eluted from the fluidic device in the washing process.
FIG. 8 is a graph illustrating effect on capture efficiency of E. coli from a urine sample as a function of urine dilution. As illustrated in FIG. 8, cell capturing efficiency was significantly increased as the E. coli-containing urine sample was more diluted. This effect was observed for both pillar array surfaces tested.

Example 7

Capturing E. coli in a Mimic Urine Solution using a Solid Substrate having a Surface with a Pillar Array: Detecting a Factor that inhibits Cell Capture

In the current example, a bacteria-containing sample was loaded to a fluidic device as described in Example 1 so that the bacterial cell was bound to the solid substrate. The number of cells in the bacteria-containing sample before and after elution through the fluidic device was determined through colony counting in order to calculate the bacteria capturing efficiency of the solid substrate.
The pillar array used had dimensions as described in Example 1. A pillar array having a SiO₂layer coated with OTC at its surface and a pillar array having a SiO₂layer coated with SAM were prepared.
As cell samples, solutions containing E. coli at 0.01 OD₆₀₀in a sodium acetate buffer or in dialyzed urine were used.
Solutions in a sodium acetate buffer (pH 4.0) used were as follows:
buffer: 100 mM sodium acetate buffer (pH 4.0);
buffer+salt: a 100 mM sodium acetate buffer (pH 4.0) solution containing 88 mM NaCl, 67 mM KCl, 38 mM NH₄Cl, and 18 mM Na₂SO₄prepared by adding 0.514 g of NaCl, 0.5 g of KCl, 0.203 g of NH₄Cl, and 0.259 g of Na₂SO₄to 100 mM sodium acetate buffer (pH 4.0);
buffer+salt+urea: a solution that consists of the “buffer+salt” solution and 333 mM urea;
buffer+salt+urea+creatine: a solution that consists of the “buffer+salt” solution, 333 mM urea, and 9.8 mM creatine;
buffer+salt+urea+creatine+uric acid: a solution that consists of the “buffer+salt” solution, 333 mM urea, 9.8 mM creatine, and 2.5 mM uric acid; and
buffer+salt+urea+creatine+uric acid+glucose: a solution that consists of the “buffer+salt” solution, 333 mM urea, 9.8 mM creatine, 2.5 mM uric acid, and 0.6 mM glucose.
An E. coli-containing solution in each of the above sodium acetate buffer (pH 4.0) solutions was obtained by diluting 10 μL of an E. coli sample in LB medium (OD₆₀₀=1.0) with 990 μL of the appropriate sodium acetate buffer (pH 4.0) solution.
The E. coli solutions in dialyzed urine were obtained as follows. First, dialyzed urine was mixed with a 2× version of each of the above sodium acetate buffer solutions at a 1:1 ration, resulting in a final pH of 3.97. Then, an E. coli-containing solution in each of the dialyzed urine solutions was obtained by diluting 10 μL of an E. coli sample in LB medium (OD₆₀₀=1.0) with 990 μL of the appropriate dialyzed urine solution.
200 μl of the E. coli sample was pumped through the fluidic device at a flow rate of 200 μl/minute. This experiment was repeated three times for each E. coli sample. The number of cells in the bacteria-containing sample before and after elution through the fluidic device was determined through colony counting in order to calculate the bacteria capturing efficiency of the solid substrate.
FIG. 9 is a graph illustrating capturing efficiency results for the various E. coli samples using the solid substrate having a surface with a pillar array. As illustrated in FIG. 9, the capturing efficiency was affected by the composition of the E. coli sample. For example, the capturing efficiency was lower in each “dialyzed urine” sample as compared to the corresponding sodium acetate buffer sample and capturing efficiency was lowered with increasing concentration of the salt contained in the E. coli sample (compare the buffer and “buffer+salt” columns for the sodium acetate samples or for the dialyzed uring samples). Other materials in the E. coli samples, such as creatine, also affected the capturing efficiency in the sodium acetate buffer samples at these conditions.

Example 8

Capturing E. coli in Urine using a Solid Substrate having a Surface with a Pillar Array: Capturing Efficiency as a Function of Dilution Rate of Urine and Flow Rate

In the current example, a bacteria-containing sample was loaded to a fluidic device as described in Example 1 so that the bacterial cell was bound to the solid substrate. The number of cells in the bacteria-containing sample before and after elution through the fluidic device was determined through colony counting in order to calculate the bacteria capturing efficiency of the solid substrate.
A pillar array having a SiO₂layer coated with PEIM by SAM coating process at its surface prepared. The pillar array had dimensions as described in Example 1.
To prepare samples, 100 mM sodium acetate buffer was mixed with urine in the desired dilution ratios until the final volume of the solution was 1 ml. Then, 10 μl of 1.0 OD₆₀₀ E. coli was added to the diluted urine solution.
200 μl of the E. coli sample in diluted urine was pumped through the fluidic device at one of three different flow rates between 100 and 500 μl/minute. This experiment was repeated for three different flow rates for each dilution ratio of the urine solution: 100, 300, and 500 μl/minute. The number of cells in the bacteria-containing sample before and after elution through the fluidic device was determined through colony counting in order to calculate the bacteria capturing efficiency of the solid substrate.
FIG. 10 is a graph illustrating capturing efficiency results for E. coli contained in the diluted urine sample using the solid substrate having a surface with a pillar array as a function of urine dilution ratio and flow rate. As illustrated in FIG. 10, as dilution of the urine sample increased, the cell capturing efficiency was increased; on the other hand, at a given urine dilution ratio, as the flow rate increased, the cell capturing efficiency decreased.
According to the method of separating a cell from a sample disclosed herein, a cell, such as a bacterial cell, fungus cell, or virus, present in a biological sample, can be efficiently separated.
By using the device for separating a cell from a sample disclosed herein, a cell, such as a bacterial cell, fungus cell, or virus, present in a biological sample, can be efficiently separated.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The terms “a” and “an” do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. The term “or” means “and/or”. The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”).
Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable.
All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein. Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

1. A method of separating a microorganism cell from a sample comprising

contacting a nonplanar solid substrate with a sample, wherein the sample comprises a cell and a liquid medium having a pH of 3.0 to 6.0, wherein the contacting is such that cells in the sample are separated from the sample.

2. The method of claim 1, wherein the cell is a bacteria, a fungus, or a virus.

3. The method of claim 1, wherein the sample is a biological sample.

4. The method of claim 3, wherein the sample is blood, urine, or saliva.

5. The method of claim 1, wherein the sample is diluted with a phosphate buffer or an acetate buffer.

6. The method of claim 5, wherein the sample is diluted with the buffer in a ratio of 1:1 to 1:10.

7. 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.

9. The method of claim 1, wherein the nonplanar solid substrate is a solid substrate having a surface comprising a pillar structure formed of a plurality of pillars, a bead-shaped solid substrate, or a sieve-shaped solid substrate having a surface comprising pores.

10. The method of claim 9, wherein each pillar has an aspect ratio of 1:1 to 20:1.

11. The method of claim 9, wherein a ratio of pillar height to distance between adjacent pillars is in the range of 1:1 to 25:1 in the pillar structure.

12. The method of claim 9, wherein distance between adjacent pillars in the pillar structure is in the range of 5 μm to 100 μm.

13. The method of claim 1, wherein the nonplanar solid substrate is hydrophobic and has a water contact angle of 70° to 95°.

14. The method of claim 13, wherein the hydrophobic solid substrate is obtained by coating the nonplanar solid substrate with octadecyldimethyl(3-trimethoxysilyl propyl)ammonium (OTC) or tridecafluorotetrahydrooctyltrimethoxysilane (DFS).

15. The method of claim 1, wherein the nonplanar solid substrate has at least one amine-based functional group at its surface.

16. The method of claim 15, wherein the nonplanar solid substrate having at least one amine-based functional group is prepared by coating the nonplanar solid substrate with polyethyleneiminetrimethoxysilane (PEIM).

17. The method of claim 1, further comprising removing materials which are not bound to the nonplanar solid substrate with a wash buffer.

18. A device for separating a cell from a sample, the device comprising a container which comprises a nonplanar solid substrate;

a sample inlet; and

a buffer solution storage unit.

19. The device of claim 18, wherein the nonplanar solid substrate is a solid substrate having a surface comprising a pillar structure formed of a plurality of pillars, a bead-shaped solid substrate, or a sieve-shaped solid substrate having a surface comprising pores.

20. The device of claim 19, wherein each pillar has an aspect ratio of 1:1 to 20:1.

21. The device of claim 19, wherein a ratio of the pillar height to a distance between adjacent pillars is in the range of 1:1 to 25:1 in the pillar structure.

22. The device of claim 19, wherein a distance between adjacent pillars in the pillar structure is in the range of 5 μm to 100 μm.

23. The device of claim 18, wherein the nonplanar solid substrate is hydrophobic and has a water contact angle of 70° to 95°.

24. The device of claim 23, wherein the hydrophobic nonplanar solid substrate is obtained by coating the nonplanar solid substrate with octadecyidimethyl(3-trimethoxysilyl propyl)ammonium (OTC) or tridecafluorotetrahydrooctyltrimethoxysilane (DFS).

25. The device of claim 18, wherein the nonplanar solid substrate has at least one amine-based functional group at its surface.

26. The device of claim 25, wherein the nonplanar solid substrate having at least one amine-based functional group is prepared by coating the nonplanar solid substrate with polyethyleneiminetrimethoxysilane (PEIM).