KR101811134B1 - A method to detect bacteria using a stationary liquid phase lab-on-a-chip - Google Patents

Abstract

The present invention relates to a bacteria detection method using a lab-on-a-chip unit in a stationary liquid. The lab-on-a-chip unit is in the stationary liquid and includes a culture space, a cleaning space, a detection space, and a channel connecting the culture space, cleaning space, and detection space. The bacteria detection method using the lab-on-a-chip unit includes the steps of: inputting a sample in the culture space and culturing bacteria; forming a complex of captured particles, bacteria, and marker particles in the culture space; collecting the complex of captured particles, bacteria, and marker particles on the lower part of the culture space; moving the complex of captured particles, bacteria, and marker particles to the detection space from the culture space; and detecting bacteria by measuring marker particle signals in the detection space.

Description

Technical Field [0001] The present invention relates to a method for detecting bacteria using a stationary liquid phase wrap-on-a-chip,

The present invention relates to a method for detecting bacteria using a stationary liquid phase wrap-on-a-chip, and more particularly to a method for detecting bacteria by immunoassay using a stationary liquid phase wrap-on-a-chip.

Technologies for detecting analytes such as bacteria, viruses, and proteins can be applied in various fields such as disease diagnosis, food inspection, and environmental monitoring. In particular, the technology for detecting specific bacteria with high sensitivity is considered to be very important for food inspection, water quality inspection and diagnosis of diseases. More specifically, in order to prevent the food poisoning or to cope with infectious diseases in advance by checking the stability of the food in advance, a quick on-site inspection method that can be easily conducted within a few hours on site is needed.

In addition, according to the Food Sanitation Act, food safety must meet the non-detection criteria, a criterion that no foodborne pathogens should be detected in food. Therefore, in order to evaluate the safety of food, it is necessary to have a high level of sensitivity that can be detected if any one of the bacteria is present in a food sample.

Traditionally, in order to detect bacteria with high sensitivity, there is a method in which a sample is cultured on a solid medium and then the number of colonies is measured. However, since the above-described method takes at least one day, it is difficult to perform a quick inspection in the field.

Accordingly, immunoassay methods such as ELISA (enzyme-linked immunosorbent assay), methods using biosensors such as surface plasmon resonance, and lab-on-a-chip ) Have been developed. However, most of the complicated equipment is required, so it has to be tested in the laboratory, and the sensitivity to detect one bacterium has not been reached.

In recent years, there has been proposed a technique (Immunity Patent No. 10-1398764) for carrying out immunoassay by moving solid particles in a stationary liquid phase by a detection method that can be used in the field. This technique has the advantage of being applicable in the field because it uses a simple method of moving particles in a stationary liquid phase lab-on-a-chip. This method uses a capture particle in which an antibody is immobilized on a magnetic particle when used for detecting bacteria and a labeling particle in which an antibody is immobilized on a particle having no magnetic property and a labeling function Lt; / RTI >

However, even in the above-mentioned method, even if there is only one bacteria in the food sample due to the limit of the sensitivity of the analysis method and the matrix effect that the substance contained in the food sample affects the test, It was difficult to reach the sensitivity of.

KR registration No. 10-1398764

An object of the present invention is to provide a bacterial detection method using a stationary liquid phase wrap-on-a-chip capable of quickly and easily detecting a small amount of bacteria with high sensitivity using a stationary liquid phase wrap-on-a-chip.

The present invention comprises a step of incubating bacteria in a culture space using a stationary liquid phase wrap-on-a-chip comprising a culture space, a cleaning space, a detection space, and a channel connecting the culture space, the washing space and the detection space; Forming a capture particle-bacteria-bead particle complex in the culture space; Capturing the captured particle-bacteria-labeled particle complex at the bottom of the culture space; Moving the captured particle-bacteria-labeled particle complex from a culture space to a detection space; And detecting bacteria by measuring a signal of the marker particle in the detection space. The present invention also provides a method for detecting bacteria using a stationary liquid phase wrap-on-a-chip.

The present invention relates to a method for producing a culture medium, comprising: a connection part for connecting a culture tube provided on a sample space, a cleaning space, a detection space, and a channel connecting the culture space, the washing space and the detection space; Injecting a sample into the culture tube and culturing the bacteria using the stationary liquid phase wrap-on-a-chip, Forming a capture particle-bacterial-labeled particle complex in said culture tube; Bonding the culture tube to the lab-on-a-chip; Capturing the captured particle-bacteria-labeled particle complex under the sample space; Moving the capture particle-bacteria-bead particle complex from the sample space to the detection space; And detecting the bacteria by measuring a signal of the marker particle in the detection space. The present invention also provides a method for detecting bacteria by using a stationary liquid phase wrap-on-a-chip.

The method for detecting bacteria using a lab-on-a-chip according to the present invention has the effect of rapidly detecting bacteria even when a small amount of bacteria is contained, because the bacteria are cultured and detected in one apparatus.

The bacteria detection method using the lab-on-a-chip according to the present invention can be applied to a rapid on-site inspection since a bacterial culture step and a detection step are easily connected to each other and do not require complicated experimental equipment or difficult techniques.

Particularly, it can be used for food inspection, water quality inspection, disease diagnosis, and the like which detect specific bacteria with high sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a view showing a method of detecting bacteria using a stationary liquid phase wrap-on-a-chip containing a culture space according to an embodiment of the present invention.
2 is a drawing and a photograph showing the structure of a stationary liquid phase wrap-on-a-chip without a culture space according to another embodiment of the present invention.
3 is a diagram illustrating a method of detecting bacteria using a stationary liquid phase wrap-on-a-chip without a culture space according to another embodiment of the present invention.
4 is a photograph of a lab-on-a-chip fabricated in an embodiment of the present invention.
5 is a photograph showing a culture tube connected to a lab-on-a-chip produced in an embodiment of the present invention.
FIG. 6 is a graph showing that the absorbance increases in proportion to the number of Vibrio parahaemolyticus contained in a sample when cultured in a stationary liquid-phase wrap-on-a-chip according to an embodiment of the present invention.
FIG. 7 is a graph showing that the absorbance increases in proportion to the number of Vibrio parahaemolyticus contained in a sample when cultured in a culture tube according to an embodiment of the present invention and then detected by being connected to a stationary liquid phase wrap-on-a-chip.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. Prior to this, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor should appropriately interpret the concepts of the terms appropriately It should be interpreted in accordance with the meaning and concept consistent with the technical idea of the present invention based on the principle that it can be defined.

Therefore, the embodiments described in this specification and the configurations shown in the drawings are merely the most preferred embodiments of the present invention and do not represent all the technical ideas of the present invention. Therefore, It is to be understood that equivalents and modifications are possible.

FIG. 1 is a schematic view showing a method of detecting bacteria using a stationary liquid-phase wrap-on-a-chip in which a culture space is included according to an embodiment of the present invention. FIG. FIG. 3 is a view showing a bacteria detection method using a stationary liquid phase wrap-on-a-chip without a culture space according to another embodiment of the present invention. FIG. Fig. 5 is a photograph showing a culture tube connected to a lab-on-a-chip manufactured in the embodiment of the present invention. Fig. 6 is a photograph of a lab-on-a- FIG. 7 is a graph showing that the absorbance increases in proportion to the number of Vibrio parahaemolyticus contained in the sample when the sample is detected without culture in the culture tank, And the absorbance increases in proportion to the number of Vibrio parahaemolyticus contained in the sample when the sample is incubated in a stationary liquid phase wrap-on-a-chip.

Hereinafter, the bacteria detection method using the stationary liquid phase wrap-on-a-chip according to the present invention will be described in detail with reference to FIGS. 1 to 7 and Examples.

The present invention relates to a method for detecting bacteria using a stationary liquid phase wrap-on-a-chip.

The term "lab-on-a-chip" used in the present invention refers to a chip manufactured to implement various operations such as mixing, reaction, separation, and analysis performed in a laboratory. Means that the analysis can be performed by moving the solid phase instead of moving the liquid phase in the main analysis step, unlike the conventional method of moving the liquid phase.

As used herein, the term "bacteria " refers to a very small unicellular organism and refers to conventional bacteria.

Hereinafter, with reference to FIGS. 1 and 2, a method for detecting bacteria using the stationary liquid phase wrap-on-a-chip according to the present invention will be described in detail.

The stationary liquid phase wrap-on-a-chip according to an embodiment of the present invention is composed of a culture space, a cleaning space, and a detection space, and comprises channels connecting the culture space, the cleaning space, and the detection space.

As used herein, the term "culture space" means a space in which bacteria are cultured, and "washing space" means a space formed between the culture space and a detection space, Means a space which provides for the washing to occur while the bacterial-labeled particle complex passes from the culture space to the detection space. In another aspect, this means a space formed between the sample space and the detection space to provide a catch particle-bacteria-display particle composite to be described later to be washed while passing from the sample space to the detection space.

In addition, the term "detection space" means a space containing a detection solution for detecting bacteria, and "sample space" means a space containing a mixed solution of a sample.

On the other hand, in the stationary liquid phase wrap-on-a-chip of the present invention, the channel is filled with a hydrophobic liquid. Here, the term "hydrophobic" refers to a property opposite to hydrophilicity and having no affinity for water, and may mean a property not to be mixed with water.

For example, the channel may be filled with a liquid that is immiscible with water, such as olive oil, mineral oil, liquid wax, and the like.

The liquid not only prevents the solutions in different spaces from mixing with each other, but also cleanses and removes impurities when the trapped particle-bacteria-labeled particle complex passes through the channel. On the other hand, it is preferable that the hydrophobic liquid has a predetermined viscosity in order not to move easily to the neighboring space.

As a specific example, when a material such as a liquid wax which exists in a solid state in a refrigerated condition and exists in a liquid state at a room temperature is filled in the channel and stored in a refrigerator, the effect of reliably separating different spaces of the lab- .

For example, the hydrophobic liquid may be a solid state at a temperature of 10 to 20 DEG C or lower, and a liquid state at a temperature of 10 to 20 DEG C or higher.

The bacteria detection method using the stationary liquid phase wrap-on-a-chip of the present invention uses a stationary liquid phase wrap-on-a-chip composed of a culture space, a cleaning space, a detection space and channels connecting the culture space, the cleaning space and the detection space, Adding a sample to the culture medium and culturing the bacteria; Forming a capture particle-bacteria-bead particle complex in the culture space; Capturing the captured particle-bacteria-labeled particle complex at the bottom of the culture space; Moving the captured particle-bacteria-labeled particle complex from a culture space to a detection space; And detecting bacteria in the detection space by measuring a signal of the label particle; And a control unit.

Meanwhile, in the step of forming the trapped particle-bacteria-labeled particle complex, a valve may be additionally provided in a channel between the culture space and the washing space to prevent the particles from moving to the detection space. At this time, the channel may be smaller than the diameter of the culture space, the washing space, and the detection space. Such a valve may be an air valve or a conventional valve used in the art.

In particular, the culture space, the washing space, and the detection space are filled with the solution to be introduced into the space in a stationary state.

Here, the fact that the solution is stopped does not mean that the solution does not move at all but means that the analysis can be performed by moving the solid phase instead of moving the liquid phase in the main analysis step, unlike the conventional method of moving the liquid phase . The solid phase may refer to a particle or a capture particle-bacteria-bead particle complex in the present invention.

In addition, in order to prevent the capture particles or the labeled particles from moving toward the detection space due to the random movement of the particles in the step of culturing the bacteria or forming the capture particle-bacteria-labeled particle complex in the culture step, The lab-on-a-chip can be kept tilted so that the space is higher.

On the other hand, in the case of using fluorescently labeled particles, instead of providing a valve, the culture space, the washing space, the detection space and the channels of the lab-on-a-chip are all filled with a culture medium as shown in Fig. 2, On-the-chip may be vertically set up to carry the step of collecting the capture particle-bacteria-labeled particle complex. In this state, due to the action of gravity and a narrow channel, it is possible to prevent substances such as trapped particles and labeled particles from moving toward the detection space.

Further, by moving the magnet vertically to the side of the detection space from the side of the culture space in a state where the lab-on-a-chip is erected, the trapped particle-bacteria-bead particle complex can be moved to the detection space.

On the other hand, in a conventional bacterial culture step, shaking is usually applied. If the activity of the capture particle or the label particle is not impaired under the above-mentioned shaking condition, the capture particle and the label particle are put together with the sample, At the same time that the capture particle-bacteria-labeled particle complex can be formed. In this case, there is an advantage that the detection process becomes very simple.

That is, in the step of culturing bacteria, capturing particles and marker particles are injected into a sample and cultured together with the bacteria to form the capturing particle-bacteria-marker particle complex while the bacteria are growing, This has the advantage of being very simple and simple.

As a specific example, the sample used in the present invention may be diluted 10 to 500 times with a culture medium.

In one embodiment of the present invention, a culture medium diluted 100-fold can be used. This means that the sample is directly diluted by adding the culture medium without diluting it with the buffer solution.

On the other hand, the above-mentioned dilution rate is a degree that can be changed according to the application substance or purpose.

Herein, the term "culture medium" means a medium which is a conventional medium, and means that a nutrient substance required for culturing a microorganism, a plant or animal or plant is cultivated as a main component and mixed with a special purpose again.

Hereinafter, with reference to FIG. 3, a method for detecting bacteria using the stationary liquid phase wrap-on-a-chip according to the present invention will be described in detail.

According to another embodiment of the present invention, there is provided a method of manufacturing a microfluidic device, comprising: a connection part connecting a sample tube, a cleaning space, a detection space, and a channel connecting the culture space, the cleaning space, and the detection space, On-a-chip < / RTI >

More specifically, bacteria can be cultured in an additional culture tube instead of providing a culture space on the lab-on-a-chip. In this case, a sample space may be provided in place of the culture space in the stationary liquid phase wrap-on-a-chip, and a culture tube connection portion capable of coupling the culture tube to the upper portion of the sample space may be provided.

In general, when culture tubes are used, very strong shaking can be applied, thus shortening the incubation time. In addition, in the method using the culture tube, a trapping particle-bacteria-labeled particle complex can pass through the sample space, thereby allowing the captured particle-bacteria-labeled particle complex to pass through the sample space and the residue to be caught in the mesh. You may.

In the case of strong shaking conditions, the activity of the trapped particles and the labeled particles may be impaired. Therefore, it is preferable to introduce trapping particles and labeled particles after a certain period of incubation and induce complex formation under gentle shaking conditions. After incubation of the bacteria and formation of the complex, the culture tubes can be connected to the lab-on-a-chip to carry out complex capture, complex movement and detection procedures.

On the other hand, the capturing particle serving as a solid phase in the present invention can be used by immobilizing a bacterial antibody on magnetic particles. When magnetic particles are used, the movement of trapped particles can be easily controlled by using a magnet.

In addition, the labeling particle can be used by immobilizing a bacterial antibody on a particle capable of producing a measurable signal. The signals that can be measured here include absorbance, fluorescence, luminescence, current, and magnetic force.

More specifically, when magnetic particles are used as trapping particles, the label particles can be used by immobilizing an enzyme and an antibody such as horseradish peroxidase (HRP) on silica particles having no magnetic property. HRP activity can be measured by absorbance, fluorescence, It can be measured in various ways. The reason that the marker particles should be free of magnetism is to exclude labeled particles that are not bound to the bacteria when the capture particle-bacteria-marker particle complex is collected by the magnet.

It is also possible to immobilize the antibody on the fluorescent particles having no magnetism and use it as the labeled particles. Fluorescent materials are generally less environmentally sensitive than enzymes and less likely to change during storage. Particularly, fluorescent materials made of transition metal ions such as europium have high stability and high excitation wavelength and emission wavelength. Therefore, when the antibody is immobilized on the europium fluorescent particles, it can be used as a labeled particle to detect a high sensitivity.

As a specific example, the sample used in the present invention may be diluted 10 to 500 times with a culture medium.

In one embodiment of the present invention, a culture medium diluted 100-fold can be used. This means that the sample is directly diluted by adding the culture medium without diluting it with the buffer solution.

Herein, the term "culture medium" means a medium which is a conventional medium, and means that a nutrient substance required for culturing a microorganism, a plant or animal or plant is cultivated as a main component and mixed with a special purpose again.

On the other hand, when bacteria are detected from a solid sample, generally a method of preparing a sample by adding a dilution buffer solution or distilled water at a ratio of 10 ml of a dilution buffer solution per gram of a solid sample is used.

There was no problem when the samples thus prepared were cultured in a solid medium as in the method for measuring the number of colonies, or cultured in a liquid medium for a long time or longer. However, when the sample thus prepared is added to a liquid medium, the culture medium is diluted and the growth rate of the bacteria is slowed, so that there may be a difference of 1 hour or more before reaching the measurable number of bacteria. Therefore, when rapid detection is required for the purpose of the present invention, a direct culture medium can be used instead of using dilute buffer solution or distilled water for preparing samples.

In addition, in the step of culturing the bacteria, the present invention can form the captured particle-bacteria-labeled particle complex while the bacteria are propagated by culturing the sample together with the capture particles and the marker particles and culturing the bacteria together with the bacteria . In this case, the detection process becomes very simple and simple.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example >

Example  1. Bacterial detection using fluorescently labeled particles

Example 1-1. Raw material preparation

In this embodiment, a method of using a separate culture tubes by using a fluorescence-labeled particles Vibrio (Vibrio parahaemolyticus ) were detected.

More specifically, the trapped particles were prepared by immobilizing a Vibrio parahaemolytic antibody on magnetic particles (Amorgtec, KIMPO, Korea) having a diameter of 300 nm. Since the magnetic particles have a carboxy group on their surface, the antibody was immobilized by a chemical method using EDC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride].

The labeled particles were prepared by immobilizing Vibrio parahaemolytic antibodies in 300 nm diameter europium fluorescent particles (Thermo Scinetific, USA). Since europium fluorescent particles also have carboxy on the surface, the antibody was immobilized by a chemical method using EDC and N-hydroxysuccinimide.

Nutrient broth (Difco) supplemented with 3% NaCl was used for the culture of Vibrio parahaemolyticus. Antibodies against Vibrio parahaemolyticus were purchased from Aptlon (Korea).

Examples 1-2. Lab-On-Chip Production

Vibrio enteritis parahaemolyticus ) was prepared by PDMS as shown in FIG. 4, and a 1 mm thick glass plate was attached to the bottom of the body. An 8 mL vial was used for the culture tube, and an acrylic wrap-on-a-chip case was prepared so as to connect the culture tube (FIG. 5).

On the other hand, the lab-on-a-chip chip is provided with a hole with a screw tab so that the vial can be fixed to a portion where the upper end of the sample space is located. Solution B (0.25% BSA in PBS).

Examples 1-3. Bacterial detection

The absorbance was measured by absorbance-viable cell count, and the number of Vibrio parahaemolyticus was confirmed and diluted to 0.20, 200, 2000, 20000, 200000 CFU / ml using a liquid culture medium. At this time, 50 μl of each sample was used to include 0, 1, 10, 100, 1000, and 10000 CFU.

50 占 퐇 of bacteria, 20 占 퐇 of 0.25% buffer solution B, 10 占 퐇 of 1 mg / ml capturing particles and 10 占 퐇 of 1 mg / ml labeled particles were placed in a 200 占 퐇 tube and reacted at room temperature for 20 minutes. After the reaction, the sample was placed in the sample space, and the magnet was placed under the sample space to collect the captured particle-bacteria-bead particle complex for 2 minutes.

Then, the magnet under the lab-on-a-chip was moved to the detection space at a speed of 30 mm / min while vibrating the lab-on-a-chip. In particular, the process of collecting and moving the trapped particle-bacteria-labeled particle complex was performed by inclining the lab-on-a-chip at a 10 degree angle with respect to the horizontal plane so that the height of the detection space was higher than that of the sample space.

Thereafter, the fluorescence of the detection space was measured using a plate reader under the conditions of Excitation 360 nm and Emission 620 nm.

As a result, as shown in Fig. 6, it was confirmed that a correlation was observed according to the number of bacteria from 100 or more Vibrio parahaemolyticus.

Example 2 Detection of Bacteria Using Red Spot Sample

In this example, an experiment was conducted to measure Vibrio parahaemolyticus by a culture method in a red sea bream sample.

More specifically, 25 g of the red ginseng sample was crushed by adding it to 225 ml of the medium, and then 90 ml of the medium was added to 10 ml of the crushed lysate to prepare a 100-fold diluted sample solution. The final concentrations of the enteric vibrio fits were 0, 1, 10 and 100 CFU / To prepare a test sample.

2 ml of each test sample was put into a sterilized 8 ml vial and incubated at 37 ° C for 2 hours and 30 minutes with shaking at 250 rpm. 10 μl of 1 mg / ml capturing particles and 10 μl of 1 mg / ml labeled particles were added and cultured at 37 ° C for 30 minutes with shaking at 150 rpm. The culture vial was connected to the lab-on-a-chip to collect the capture particle-bacteria-bead particle complex as described in Example 1 and then moved to the detection space. Finally, the fluorescence of the detection space was measured using a plate reader at Excitation 360nm and Emission 620nm.

As a result, as shown in Fig. 7, it was confirmed that even if only 2 germs were detected in 2 ml of a sample having a concentration of 1 cfu / ml, detection was possible. Here, the experiment was carried out under the condition that two bacteria were contained per each sample, but it is expected that even if only one germ can be detected, it can be detected in consideration of the difference from the sample without bacteria.

The total time required for the detection was 3 hours and 30 minutes including the incubation time. From the results of the present example, the detection method of the present invention can detect a single bacteria contained in the sample with a simple method on the spot .

While the present invention has been particularly shown and described with reference to the specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible, will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

Using a stationary liquid phase wrap-on-a-chip consisting of a culture space, a cleaning space, a detection space and a channel connecting the culture space, the cleaning space and the detection space,
Placing a sample in the culture space and culturing the bacteria;
Forming a capture particle-bacteria-labeled particle complex using capture particles immobilized with a bacterial antibody on magnetic particles and non-magnetic marker particles in the culture space;
Capturing the captured particle-bacteria-labeled particle complex at the bottom of the culture space using a magnet outside the culture space;
Moving the magnet from the outside of the incubation space to the detection space and moving the trapped particle-bacteria-bead particle complex from the incubation space to the detection space; And
And detecting the bacteria by measuring a signal of the indicator particle in the detection space. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;
A connection part for connecting the culture tube provided on the upper part of the sample space, the connection part comprising a sample space, a cleaning space, a detection space, and a channel connecting the sample space, the cleaning space, and the detection space; On-a-chip &lt; / RTI &gt;
Injecting a sample into the culture tube and culturing the bacteria;
Forming a capture particle-bacteria-bead particle complex using capture particles immobilized with a bacterial antibody on magnetic particles and non-magnetic beacon particles in the culture tube;
Bonding the culture tube to the lab-on-a-chip;
Capturing the captured particle-bacteria-labeled particle complex under the sample space using a magnet outside the sample space;
Moving the magnet from the sample space to the detection space and moving the capture particle-bacteria-bead particle complex from the sample space to the detection space; And
And detecting the bacteria by measuring a signal of the indicator particle in the detection space. &Lt; RTI ID = 0.0 &gt; 15. &lt; / RTI &gt;
The method according to claim 1,
And a valve is additionally provided between the culture space and the cleaning space.
3. The method according to claim 1 or 2,
The channel
Characterized in that it is filled with a hydrophobic liquid.
5. The method of claim 4,
The hydrophobic liquid
Wherein the wax is in a solid state in a refrigerated condition and is present in a liquid state in a room temperature.
The method according to claim 1,
The label particles
The bacterial antibody is immobilized on the fluorescent particles,
Wherein the culture space, the washing space, the detection space and the channel are filled with a culture medium.
The method according to claim 1,
The step of culturing the bacteria
On-the-fly chip is subjected to the bacterial culture in a vertical state so that the detection space is located at an upper position.
3. The method according to claim 1 or 2,
The sample
A method for detecting bacteria using a lab-on-a-chip, wherein the bacteria is diluted 10 to 500 times with a culture medium.
3. The method according to claim 1 or 2,
The step of culturing the bacteria
Wherein said capture particle and said labeling particle are injected into said sample and said bacteria are cultured to form said capture particle-bacteria-label particle complex while said bacteria are growing. Way.
The method according to claim 1,
The step of forming the capture particle-bacterial-labeled particle complex
On-the-fly chip is tilted so that the detection space is positioned higher than the incubation space.
3. The method of claim 2,
The step of forming the capture particle-bacterial-labeled particle complex
On-the-fly chip is tilted so that the detection space is located higher than the sample space.
3. The method of claim 2,
The sample space
Characterized in that a mesh is additionally provided so that only the captured particle-bacteria-labeled particle complex is selectively transferred from the culture tube to the sample space.

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