KR101722997B1 - Integrated device enabling biomolecule analysis and method using centrifugal force - Google Patents

Abstract

The present invention relates to an integrated biomolecule analyzing apparatus, and more particularly, to a centrifugal force-based integrated biomolecule analyzing apparatus capable of temperature control, gene extraction, gene amplification, cell culture, and gene detection simultaneously.
The integrated biomolecule analyzing apparatus according to the present invention can be carried out integrally with gene extraction, gene amplification and gene detection. Thus, the problem of performing gene extraction, gene amplification, or gene detection separately in gene detection has been improved. In addition, since the integrated biomolecule analyzing apparatus according to the present invention can detect a gene in a short time using a simple and automated method, it is possible to immediately determine whether or not the microbes causing diseases such as food poisoning occur in the restaurant or the food industry And can be used for the detection of various diseases that require prompt and on-site diagnosis.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a biomolecule-

The present invention relates to an integrated biomolecule analyzing apparatus, and more particularly, to a centrifugal force-based integrated biomolecule analyzing apparatus capable of temperature control, gene extraction, gene amplification, cell culture, and gene detection simultaneously.

In general, when a disease occurs, it will work to scientifically identify its cause. In particular, it is the most common method to count the number of cells directly by collecting a sample from a disease occurrence site, culturing the cell, and dispersing it on a substrate. Recently, it is possible to determine whether a pathogen or a virus causing a specific disease is infected by amplifying the gene of the cultured cell, detecting the amplified gene, immunoassay, fluorescence energy transfer analysis, microarray analysis Are being developed.

However, all of these detection methods are very cumbersome because they are performed separately at each step in the laboratory. In addition, moving the samples to the laboratory and separating them in stages will take a long time, so that the disease is spreading rapidly and rapidly, and it is already late to respond appropriately to the disease.

For example, food poisoning is known to be caused by various causes such as Norovirus, Salmonella, Escherichia coli, and Staphylococcus aureus, and its transmission rate is also very fast in summer. This food poisoning is caused by a lack of diagnostic tools that can not be distinguished at the restaurant or catering industry, where samples are transferred to the laboratory to cultivate the cells and count the number of cultured cells to determine whether the pathogens are infected. Also, since it takes up to 7 days to confirm the results, food poisoning is already spreading at a high speed and it is too late to take proper action.

In order to overcome the disadvantages of conventional discrimination methods requiring such a long period, recently, methods for diagnosing the occurrence of the disease within a day through nucleic acid amplification, fluorescence energy transfer, enzyme immunoassay and microarray have been developed . In this case, however, it is necessary to use expensive equipments and professional manpower, so that it is more suitable to be performed in the place of the laboratory unit, and the process is also complicated. Therefore, have.

The prior art document related to the present invention is disclosed in Korean Patent Laid-Open No. 10-2008-0022035 (Patent Document 1), and more specifically, a centrifugal force-based microfluidic device for detecting nucleic acid of a target cell and a microfluidic device A description related to a system is disclosed.

Patent Document 1: Korean Patent Publication No. 10-2008-0022035

SUMMARY OF THE INVENTION The present invention has been made in order to solve the above-mentioned problems, and an object of the present invention is to provide a microfluidic system using a centrifugal force based microfluidic system to collectively carry out gene extraction, amplification and gene detection And to provide an integrated biomolecule analyzing device capable of performing biomolecule analysis.

According to an aspect of the present invention, there is provided a biomolecule analyzing apparatus comprising:

On a circular plate on which centrifugal force is generated through rotation;

A reactor having a temperature control function; And

A heat transfer unit for transferring heat to the reactor;

/ RTI >

And the heat transfer part transfers the heat absorbed through the electromagnetic wave irradiation to the reactor.

Further, the reactor is characterized in performing cell culture, inter-biomolecule binding, gene extraction and gene amplification.

Further, the reactor is divided into a gene extracting unit, a gene amplifying unit, a solution separating unit, an excess solution storing unit, and a solution diluting unit including a gene, and the biomolecule analyzing apparatus further includes a gene detecting unit .

In addition, the biomolecule analyzing apparatus may be configured such that the gene extracting unit, the gene amplifying unit, the solution separating unit, the excess solution storing unit during separation, the solution diluting unit including the gene, the gene detecting unit, and the heat transfer unit are sequentially connected .

In addition, the at least one sequential connected gene extracting unit, the gene amplifying unit, the solution separating unit, the excess solution storing unit at the time of separation, the solution diluting unit including the gene, the gene detecting unit, and the heat transfer unit are formed on the circular plate, .

And the circular plate is rotated at a speed of 1,200-4,800 rpm.

The total thickness of the biomolecule analyzer is 3-6 mm.

The heat transfer unit transfers heat to the gene amplification unit through electromagnetic wave irradiation.

Further, the gene amplification unit is characterized in that at least one member selected from the group consisting of aluminum, copper, and silicon is attached to absorb the heat transmitted by the heat transfer unit.

Further, the temperature control in the reactor is performed at a temperature of 20-100 ° C.

The integrated biomolecule analyzing apparatus according to the present invention can be carried out integrally with gene extraction, gene amplification and gene detection. Thus, the problem of performing gene extraction, gene amplification, or gene detection separately in gene detection has been improved. In addition, since the integrated biomolecule analyzing apparatus according to the present invention can detect a gene in a short time using a simple and automated method, it is possible to immediately determine whether or not the microbes causing diseases such as food poisoning occur in a restaurant or a restaurant industry And can be used for the detection of various diseases that require prompt and on-site diagnosis.

1 is a diagram illustrating a plurality of centrifugal force microfluidic integrated gene detection apparatuses according to the present invention.
FIG. 2 is a diagram depicting a single integrated gene detection apparatus according to the present invention. FIG.
3 is a photograph showing a process of detecting a gene using the integrated gene detection apparatus according to the present invention.
4 is a photograph showing the result of gene detection using the integrated gene detection apparatus according to the present invention.

Accordingly, the inventors of the present invention have made intensive efforts to develop a biomolecule analyzer capable of performing gene expression, gene amplification, and gene detection integrally with each other, and as a result, discovered a biomolecule analyzer according to the present invention, .

Specifically, the biomolecule analyzing apparatus according to the present invention comprises:

On a circular plate on which centrifugal force is generated through rotation;

A reactor having a temperature control function; And

A heat transfer unit for transferring heat to the reactor;

/ RTI >

And the heat transfer part transfers the heat absorbed through the electromagnetic wave irradiation to the reactor.

Further, the reactor is characterized in performing cell culture, inter-biomolecule binding, gene extraction and gene amplification.

In addition, the reactor is divided into a gene extracting unit 1, a gene amplifying unit 2, a solution separating unit 3, an excess solution storing unit 4 during separation, and a solution diluting unit 5 including a gene And the biomolecule analyzer further comprises a gene detector (6).

In the gene extracting unit, a sample suspected of causing a disease is injected to extract a gene from a causative organism or virus for the disease. Although there is no particular limitation in this case, it is preferable to mix the sample solution suspected of being infected with the causative bacteria of the disease and the virus and the solution containing the magnetic beads in which the antibody capable of binding specifically to the causative bacteria and the virus gene is immobilized, From the volumetric sample solution, the bacteria to be detected can be selectively concentrated into small volumes suitable for extraction. Preferably, the solution is mixed for 1-30 minutes, and then the mixture is injected into the gene extracting section of the reactor. As a gene extraction method, a thermal extraction method may be utilized by irradiating electromagnetic waves, and the magnetic beads preferably serve as a heat transfer body.

The gene amplification unit amplifies the gene extracted from the gene extraction unit. The amplification method at this time is not particularly limited, but it is preferable to use Recombinase Polymerase Amplification (RPA). Also, although there is no particular limitation, the temperature for amplification is preferably 37-40 ° C. If the amplification temperature is lower than 37 ° C, it is not preferable because it does not provide a sufficient temperature for amplification. ° C., the enzymes for amplification cause heat denaturation and the like, which is not preferable. Also, although there is no particular limitation, it is preferable that the amplification time is 17-23 minutes. If the amplification time is less than 17 minutes, sufficient amplification can not be performed and reliability of the result can be deteriorated. If the time exceeds 23 minutes, the probability of obtaining a false negative error due to nonspecific binding increases, and it takes an unnecessary time even if sufficient amplification is performed.

The solution separating unit separates the amplified gene solution as much as necessary for detection.

Thus, the solution is separated into the volume required for detection by the solution separator, and the excess capacity is separately stored in the excess solution reservoir upon separation.

Also, the solution containing the gene separated and amplified by the solution separating unit is separated by the solution separating unit, and then moved to the solution diluting unit containing the gene capable of diluting the solution. There is no particular limitation on the dilution part of the solution containing the gene, but a dilution solution is preferably included, and the dilution solution can be used without any particular limitation as long as it is a solvent used for dilution.

In addition, the gene detecting unit detects a gene using a solution that has passed through a solution dilution unit containing the gene. Gene detection is not limited, but is detected using a lateral flow sensor. The gene detection section may include a threshold structure 8 to prevent dispersion of components required for detection when the diluted solution is transferred using centrifugal force and the lateral flow sensor has absorbed the diluted solution. In addition, since the portion of the membrane in which the dilute solution and the components are moved can be inhibited when the membrane is in contact with the structure, the partial cutting structure 9 can be selectively lifted without touching the membrane portion .

Further, the heat transfer part transmits heat to the gene amplification part to enable smooth gene amplification. The method of transferring the heat by the heat transfer part is not particularly limited, but may be heat transfer by electromagnetic wave irradiation. In addition, a portion where the electromagnetic wave is irradiated by the heat transfer portion may be irradiated to the vicinity of the gene amplification portion to transmit heat. To this end, a metal thin film is further provided in the vicinity of the gene amplification portion, . ≪ / RTI > In the case of transferring heat by the electromagnetic wave irradiation, it is possible to transmit heat in a more elaborate and stable manner than in the case of transferring heat by other heat supply sources, and the temperature can be more easily controlled. Further, since the metal thin film is preferably made of a metal having excellent thermal conductivity for the purpose of delicate heat transfer and temperature control, it is preferable that the metal thin film is composed of at least one of aluminum, copper and silicon. In addition, when the metal thin film is irradiated with electromagnetic waves to transmit heat to the cell culture unit and the gene amplification unit, it is possible to contribute to the miniaturization and the ultra-light weight of the whole apparatus rather than the method of supplying heat by other heat supply sources. Thereby contributing greatly to the portability of the integrated gene detection apparatus.

On the other hand, the temperature control in the reactor is preferably performed at a temperature of 20-100 ° C.

Meanwhile, the biomolecule analyzing apparatus according to the present invention is characterized in that the biomolecule analyzing apparatus according to the present invention is characterized in that the biomolecule analyzing apparatus according to the present invention is characterized in that the gene extracting unit, It is preferable that they are connected. At this time, the diameter of the flow channel is not particularly limited, but is preferably 0.5? 1 mm.

The sequencially connected gene extractor, the gene amplification unit, the solution separator, the excess solution reservoir during separation, the solution diluting unit including the gene, the gene detector, and the heat transfer unit may be composed of one or more than one on the circular plate.

Further, the biomolecule analyzing apparatus can automate the process of detecting a gene using the centrifugal force generated when the circular body rotates. At this time, it is preferable that the rotation speed of the circular body for generating the centrifugal force is 1200-4800 rpm. If the rotation speed is less than 1200 rpm, it is difficult to draw the smooth flow of the solution and the rotation speed is 4800 rpm The centrifugal force more than necessary may be generated to increase the power consumption, and the system may become unstable due to vibration or the like, which is not preferable.

The sequential connection by the flow channels of the gene extraction unit, the gene amplification unit, the solution separation unit, the excess solution storage unit during separation, the solution diluting unit including the gene, the gene detection unit, and the heat transfer unit, It is preferable to allow the microfluid to move more efficiently.

Also, the connection of the channels may include a gene extracting unit, a gene amplifying unit, a solution separating unit, an excess solution storing unit in separation, a solution diluting unit including a gene, a gene detecting unit, and a heat transferring unit, starting from the center of the circular body, It is preferable to sequentially connect them by the flow channel because the centrifugal force can be utilized more efficiently. That is, when sequentially starting from the center of the circle and connecting outwardly, it is preferable that the gene can be detected while sequentially moving the solution by each region by centrifugal force without any special external force.

The total thickness of the biomolecule analyzer including the gene extraction unit, the gene amplification unit, the solution separation unit, the excess solution storage unit during separation, the solution dilution unit including the gene, the gene detection unit, and the heat transfer unit provided on the circular plate is 3- 6 mm. If the thickness is less than 3 mm, it is not preferable since sufficient volume of each storage part for gene detection can not be ensured. If the thickness exceeds 6 mm, the biomolecule analysis It is not preferable because the portability of the apparatus is restricted.

The biomolecule analyzing apparatus according to the present invention configured as described above is capable of integrally performing gene extraction, gene amplification, and gene detection while using centrifugal force. Such a biomolecule analyzer is excellent in portability, so that it is possible to detect a gene immediately in a site where a disease occurs, and it is possible to identify the cause and occurrence of the disease on the spot. In case of a disease which must be treated immediately, such as food poisoning due to rapid infectivity, the biomolecule analyzing apparatus according to the present invention enables immediate gene detection and disease occurrence discrimination in a restaurant or a food industry field.

The method for diagnosing a disease according to another aspect of the present invention is diagnosed using the biomolecule analyzing apparatus according to the present invention. The disease diagnosed by the diagnostic method is not particularly limited. However, it may preferably be any one selected from the group consisting of food poisoning, vomiting, sepsis, acquired immunodeficiency, and the like.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Example

A biomolecule analyzer as shown in Fig. 1 was prepared. FIG. 2 is a view showing a single gene extraction unit, a gene amplification unit, a solution separation unit, an excess solution storage unit during separation, a solution dilution unit including a gene, a gene detection unit, and a heat transfer unit sequentially connected to a circular plate . Using these biomolecule analyzers, the genes of salmonella causing food poisoning were detected. Specifically, as a target substance for food poisoning detection, 100 μl of a magnetic bead in which an antibody specifically binding to Salmonella was immobilized was added to a sample such as milk, and the mixture was mixed for 10 minutes. After mixing, the supernatant was removed, and then 5 μl of the solution containing the magnetic beads was added to the gene extraction unit of the biomolecule analyzer. The gene is extracted from the thus-introduced solution. At this time, the electromagnetic wave is irradiated to the cell culture section for 20 seconds, and the injected magnetic bead is thermodynamically transferred to thermally extract the gene of the cultured cell (see FIG. After the extraction, the No. 1 valve was opened and the circular body disk was rotated to transfer the extracted gene solution to the gene amplification part. When the electromagnetic wave is irradiated to the gene amplification unit as shown in FIG. 3B, a metal material such as aluminum disposed on the rear surface absorbs electromagnetic waves and is heated. The temperature is kept at 40 ° C for the amplification, and heating is carried out for 20 minutes. As shown in FIG. 3C, only 10 .mu.l of the amplified solution is transferred to the separator to utilize it for detection. When the valve 2 is opened and the body is rotated, the solution is transferred to the separator. In the separator, only 10 μl of the solution containing the gene is separated to be used for detection, and the excess solution is transferred to the excess solution reservoir upon separation. 10 μl of the separated solution is transferred to the solution dilution part. At this time, the solution containing the separated gene is moved to the solution diluting part containing the gene by opening the valve 3 and rotating the body as shown in FIG. 3D. Thus, the solution containing the diluted gene is moved to the gene detecting section including the lateral flow sensor by opening the valve 4 and rotating the body as shown in FIG. 3E. Thereafter, when the body was stopped, final gene detection using a lateral flow sensor was performed to detect the Salmonella gene causing food poisoning (see FIG. 3F).

FIG. 4 is a view showing the result of performing food poisoning detection according to the present embodiment. Specifically, FIG. 4 is a photograph of a food-borne pathogen (Salmonella) detected using a biomolecule analyzer according to the present embodiment. The total detection time was about 30 minutes.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. It is natural.

1. The gene extraction unit
2. Gene amplification unit
3. Solution separator
4. Excess solution storage part
5. Dilution of the solution containing the gene
6. Gene detection unit
7. Heat transfer part
8. Threshold Structure
9. Partial cutting structure

Claims (14)

A circular plate and at least one reactor arranged circumferentially on the circular plate,
The reactor includes a gene detecting unit, a gene amplifying unit, a solution separating unit, and a solution diluting unit sequentially from the center of the circular plate to the outside of the circular plate, and connected to a lower portion of the solution diluting unit, Wow,
An excess solution storage part formed on the lower surface of the gene amplification part so as to be separated from and connected to the solution separation part,
A flow connecting the gene amplification unit and the gene amplification unit, the gene amplification unit and the solution separation unit, the gene amplification unit and the excess solution storage unit, the solution separation unit and the solution dilution unit, and the solution dilution unit and the gene detection unit, Channel,
And a heat transfer unit attached to the gene amplification unit and absorbing heat due to the irradiated electromagnetic wave and transmitting the heat to the gene amplification unit,
Wherein the heat transfer portion is at least one selected from the group consisting of aluminum, copper, and silicon, and the electromagnetic wave is irradiated to the gene amplification portion,
Wherein the gene detecting unit comprises a lateral flow sensor for absorbing the diluted solution flowing through the solution diluting unit and displaying a mark according to presence or absence of a gene to be measured. delete delete delete delete The method according to claim 1,
Wherein the circular plate is rotated at a speed of 1,200-4,800 rpm. The method according to claim 1,
Wherein the total thickness of the biomolecule analyzer is 3-6 mm. delete delete The method according to claim 1,
Wherein the gene amplification unit is performed at 37-40 DEG C for 17-23 minutes. delete A biomolecule analyzing method using the biomolecule analyzer according to claim 1. delete delete

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