KR20190095081A - Micro bio chip for polymerase chain reaction - Google Patents

Abstract

The present invention relates to a micro-biochip. More specifically, the present invention relates to the micro-biochip for polymerase chain reaction (PCR), in a molecular diagnosis using PCR, conducts a reaction of a single sample with different reagents to diagnose various diseases, but by using the action of a hydrophilic-treated surface of a sealing film attached to the lower surface of a substrate, the sample injected into a single sample injection chamber without power is transferred to a plurality of reagent storage chambers. Therefore, it is not necessary to store the sample, has a simple chip structure, and does not need a separate power source, thereby being able to be easily used in the filed as well as being able to prevent the sample from being contaminated since the sample is transferred through a plurality of mixed channels, so that reliability of the single sample is improved to enable multiple diagnosis.

Description

Micro biochip for PCR {MICRO BIO CHIP FOR POLYMERASE CHAIN REACTION}

The present invention relates to a micro-biochip, and more particularly, in a molecular diagnosis using a polymerase chain reaction (PCR), a single sample is reacted with different reagents to diagnose various diseases, and a single force is used. The sample injected into the sample injection chamber is transferred to multiple reagent storage chambers through multiple mixing channels, so it is not necessary to store the sample, so it is easy to use in the field and the sample is prevented from being contaminated, thus improving the reliability of a single sample. The present invention relates to a microchip biochip for enabling multiple diagnosis.

Molecular diagnostics is an in vitro diagnostic method that detects the causes of infection and infection by detecting nucleic acids (DNA and RNA or variants thereof) such as bacteria and viruses that cause diseases.

This molecular diagnosis process consists of three major steps, in order to obtain the pure DNA or RNA in the cell, the pre-treatment process, gene amplification process using polymerase chain reaction (PCR), and analysis of the amplification products. It is a process of detecting whether or not.

In other words, the gene must be amplified in order to detect the cause of the disease or the presence of an infection using a very small amount of the gene. Polymerase Chain Reaction (PCR) is a method of repeatedly heating and cooling genetic material to serially replicate a region having a specific sequence of a nucleic acid to exponentially amplify the genetic material having a specific sequence region. It is a technology that can.

DNA replication process through polymerase chain reaction is carried out through the step-by-step replication process as follows. In other words, PCR begins with double-stranded DNA, and the first reaction of each cycle is the separation of two strands through heat treatment. This process is called 'denaturing' and is usually performed at 94 ° C. Next is the cooling process, which heterologizes the complementary sequence of two DNA strands with primers separated, which is called 'annealing' and is run at 55 ° C. The final step is the polymerization process, where the DNA polymerase in the mixture starts DNA synthesis from two primers using four deoxyribonucleotides. This process is called 'extension' Will be executed.

This in vitro DNA amplification process, the PCR process, is considered to be the most suitable biochemical analysis process for miniaturizing the entire experimental system because of the simplicity of the experimental step and the simple configuration of additional devices for the experiment. It can reduce the consumption of the sample used in the experiment, and it can be used not only in the laboratory but also in the field because it can not only shorten the whole experiment time with the fast heating rate and cooling rate due to the much smaller heat capacity.

In this field investigation, micro biochips are used to transfer and react a small amount of fluid. The micro biochip is provided with a chamber for trapping a small amount of fluid, a channel through which the fluid can flow, and a valve means for controlling the flow of the fluid. The biochip is made of glass, silicon or plastic to form a plurality of chambers and channels on the substrate to enable field investigation.

On the other hand, the biochip needs a driving pressure for transferring or mixing the fluid in the chamber. As such a driving pressure, capillary pressure may be used, and the pressure by a separate pump may be used. Recently, disc-shaped biochips that drive fluid by centrifugal force by rotating disc-shaped biochips having chambers and channels arranged therein have been proposed.

Figure 6 shows a biochip 100 capable of metering according to the prior art. As shown, the conventional biochip 100 includes a substrate 109 having a constant size, a plurality of chambers formed in the substrate 109 to receive a fluid, and a plurality of channels for transporting the fluid in the chamber. It includes. For example, the plurality of chambers may include a sample storage chamber 110 for injecting and receiving a sample, a reagent storage chamber 120 for injecting and receiving a reagent, and a mixing channel 130 for mixing a sample and a reagent. ) The plurality of channels include a sample transfer channel 111 for transferring a sample in the sample storage chamber 110 and a reagent transfer channel 121 for transferring a sample in the sample storage chamber 120.

Therefore, when using such a biochip 100, a predetermined amount of reagent is injected into the plurality of reagent storage chambers 120 through a plurality of reagent inlets 125 using a pipette or the like. At this time, since different types of reagents are often injected into the reagent storage chamber 120, it is preferable for a skilled person to separately inject reagents in a hospital or a laboratory. In the field, the collected samples are injected into the plurality of sample storage chambers 110. That is, the biochip 100 is laid horizontally, and the sample is injected through any one sample inlet 115 using a pipette to two sample inlets 115. Then, the sample injected into any one sample storage chamber 110 is moved to the neighboring sample storage chamber 110 through the channel 112 for quantification. The sample is injected until all sample storage chambers 110 are filled. As such, it is easy to inject the sample to the plurality of sample storage chambers 110 in a quantitative manner so that an unskilled person can easily quantitate the sample in a poor environment.

In addition, samples injected into the plurality of sample storage chambers 110 are stored in the sample storage chambers 110. That is, since the sample transfer channel 111 formed in the sample storage chamber 110 has a very small cross-sectional area, the sample transfer channel 111 functions as a valve to prevent the sample in the sample storage chamber 110 from moving to the reagent storage chamber 120. Therefore, the biochip 100 into which both the sample and the reagent are injected may mix the sample and the reagent by using a centrifuge.

As such, the conventional micro biochip has a problem in that it is inconvenient to use in the field and requires expensive equipment because a separate external power such as a centrifuge or a pump must be used to mix the sample and the reagent.

In addition, in the conventional micro biochip, since samples are stored in a plurality of sample storage chambers for a predetermined time, foreign substances may be introduced or other kinds of drugs may be mixed during the storage process. Therefore, the conventional micro biochip has a problem that multiple diagnosis based on a single sample is difficult because it is difficult to guarantee the reliability of a single sample (same one sample) transferred to a plurality of reagent storage chambers and mixed channels. .

Republic of Korea Patent No. 10-1780429 (Registration Date: September 14, 2017)

In order to solve the problems of the prior art of the present invention, the main object of the present invention is to transfer a single sample from one sample storage chamber to a plurality of reagent storage chambers and mixing channels and to inject a sample into one sample storage chamber. At the same time, by allowing samples to be transferred to a plurality of reagent storage chambers and mixing channels, it is possible to provide a micro biochip for PCR that can increase the reliability of a single sample by preventing contamination of the sample during the storage of the sample in the sample storage chamber. will be.

In addition, the present invention transfers a single sample from one sample storage chamber to a plurality of reagent storage chambers and mixing channels, but when a sample is injected into one sample storage chamber, a plurality of reagents are added to the sample through a plurality of mixing channels without additional power. It is to provide a micro biochip for PCR that can be transferred to the storage chamber.

As a means for achieving the object of the present invention, the micro-biochip for PCR according to the present invention,

A substrate made of a transparent material having a predetermined thickness;

A sample injection chamber formed by penetrating the substrate up and down;

A plurality of reagent storage chambers spaced apart from the sample injection chamber by a predetermined distance and formed with grooves having a predetermined depth on a lower surface of the substrate;

A mixing channel formed by forming a channel at a predetermined depth on a lower surface of the substrate so as to connect the one sample injection chamber and the plurality of reagent storage chambers;

And a sealing film attached to a lower surface of the substrate to close the open lower ends of the one sample injection chamber, the plurality of reagent storage chambers, and the plurality of mixing channels.

The sealing film has a surface that closes the open lower ends of the one sample injection chamber, the plurality of reagent storage chambers, and the plurality of mixing channels to be hydrophilic, so that the sample injected into the one sample injection chamber is connected to the plurality of mixing channels. It is characterized in that it is quickly transferred to a plurality of reagent storage chamber through.

In the present invention, the surface of the sealing film is subjected to vacuum plasma treatment.

The sealing film is subjected to vacuum plasma treatment while attached to the lower surface of the substrate to hydrophilize the surface for closing the one sample injection chamber, the plurality of reagent storage chambers, and the plurality of mixing channels.

The sealing film is made of a polymer material including a material having excellent heat transfer rate such as nano carbon or carbon tube.

The sample injection chamber has a size such that the sample does not overflow while the injected sample is transferred to the plurality of reagent storage chambers, and a sample injection hole is formed at an upper end of the sample injection chamber, and the plurality of sample injection holes are formed on an upper surface of the substrate. A plurality of reagent inlets are formed by forming grooves of a constant depth so as to be connected to the reagent storage chamber.

The mixing channel is formed by forming a groove having a predetermined width and length on a lower surface of the substrate, the groove being connected to the reagent storage chamber and extending a predetermined length toward the sample injection chamber and having a width and height similar to that of the reagent storage chamber. And a narrow channel portion connected to the sample injection chamber and extending to a predetermined length toward the reagent injection chamber and connected to the mixing channel portion, the narrow channel portion being formed with a groove having a smaller width and height than the mixing channel. Steps are formed between the mixing channel portion and the narrow channel portion with a constant width and height.

According to the present invention, a single sample may be transferred from one sample storage chamber to a plurality of reagent storage chambers and a mixing channel while the sample may be transferred to a plurality of reagent storage chambers and a mixing channel while injecting a sample into one sample storage chamber. By preventing the sample from being contaminated during the storage of the sample in the sample storage chamber, the reliability of the single sample can be improved.

In addition, the present invention transfers a single sample from a single sample storage chamber to a plurality of reagent storage chambers and mixing channels, but when a sample is injected into one sample storage chamber, a plurality of samples using a hydrophilic surface of the sealing film without additional power Through the mixing channel of the effect can be quickly transferred to a plurality of reagent storage chamber.

In addition, since the micro biochip according to the present invention is transferred to a plurality of reagent storage chambers through a plurality of closed mixing channels immediately after injecting a sample into one sample injection chamber, the reliability of a single sample is improved, so that reliable multi-diagnosis is achieved. There is a possible effect.

1 is a perspective view showing an example of a micro biochip in the present invention,
2 is a plan view of the micro biochip shown in FIG.
3 is a cross-sectional view of the micro biochip shown in FIG.
4 and 5 is a cross-sectional view showing a method of using a micro biochip according to the present invention,
Figure 6 is a plan view showing a biochip capable of quantitative injection according to the prior art.

Advantages and features of the present invention, and a method for achieving the same will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. However, this embodiment is provided to explain in detail enough to easily implement the technical idea of the present invention to those skilled in the art. In addition, in describing the embodiments of the present invention, when it is determined that the detailed description of the related well-known configuration or function interferes with the understanding of the embodiments of the present invention, the detailed description thereof will be omitted.

1 is a perspective view showing an example of a micro biochip in the present invention, FIG. 2 is a plan view of the micro biochip shown in FIG. 1, and FIG. 3 is a cross-sectional view of the micro biochip shown in FIG.

As shown, the micro biochip 5 according to the present invention includes a substrate 51 of a constant thickness made of a transparent polymer material. The substrate 51 includes a plurality of mixing channels connecting one sample injection chamber 53 and a plurality of reagent storage chambers 55 and one sample injection chamber 53 and a plurality of reagent storage chambers 55. 54 is formed.

First, the sample injection chamber 53 is made to penetrate the substrate 51 up and down, so that a circular groove is formed at a predetermined depth on the lower surface of the substrate 51. In addition, an upper portion of the sample injection chamber 53 is formed with a sample injection opening 53a open to the top to inject a sample. Preferably, the sample injection chamber 53 and the sample injection port 53a may be formed to have the same diameter.

A plurality of reagent storage chambers 55 are formed on the bottom surface of the substrate 51. The reagent storage chamber 55 is formed to be spaced apart from a single sample injection chamber 53 by a predetermined distance. Also. The reagent storage chamber 55 has a smaller size than one sample injection chamber 53, and the sum of the plurality of reagent storage chambers 55 is smaller than the size of one sample injection chamber 53.

In addition, a plurality of reagent inlets 55a are formed at the upper ends of the plurality of reagent storage chambers 55 to inject the reagents. Preferably, the reagent inlet 55a is formed by forming a circular groove with a predetermined width and depth on the upper surface of the substrate 51. At this time, the diameter of the reagent inlet 54a is smaller than the diameter of the reagent storage chamber 55 and the depth is formed to communicate with the reagent storage chamber 55. Therefore, a stepped 55b is formed between the reagent inlet 54a and the reagent storage chamber 55 at a predetermined width.

The plurality of reagent storage chambers 55 are arranged in a row and have a size that can accommodate all of the samples injected into the one sample injection chamber 53. In addition, the sample injection chamber has a size such that the sample does not overflow while the sample to be injected is transferred to the plurality of reagent storage chambers.

In addition, a plurality of mixing channels 54 are formed between the one sample injection chamber 53 and the plurality of reagent storage chambers 55. The plurality of mixing channels 54 are formed to connect the plurality of reagent storage chambers 55 and one sample injection chamber 54 to mix the sample and the reagent.

In addition, the mixed channel 54 is divided into two parts, the mixed channel part 541 and the narrow channel part 542. The mixing channel part 541 is formed of a channel having a width and height similar to that of the reagent storage chamber 55. Preferably, the mixing channel part 54 is connected to the reagent storage chamber 55 and extends a predetermined length toward the sample injection chamber 54. The narrow channel portion 542 is formed of a channel having a smaller diameter and a higher height than the mixed channel portion 541. The narrow channel part 542 is connected to the sample injection chamber 53 and extends a predetermined length toward the reagent chamber 53 to be connected to the mixing channel part 541. In addition, the plurality of mixed channel portions 541 may be formed in a straight line, and a part of the narrow channel portions 542 may be bent or curved at a predetermined angle. Accordingly, the stepped 543 is formed between the mixed channel part 541 and the narrow channel part 542 by a height difference and a predetermined height and width.

Subsequently, a sealing film 52 is attached to the lower surface of the substrate 51. The sealing film 52 is attached to the lower surface of the substrate 51 and serves to close the open lower ends of the plurality of reagent storage chambers 55, the mixing channel 54, and one sample injection chamber 53. In addition, the sealing film 52 is made of a thin film and also serves to quickly transfer the heat and cold of the heater portion of the PCR device installed in the lower portion to the reagent storage chamber 55 and the mixing channel 54. To this end, the sealing film 52 may be made of a metal or a polymer material having excellent heat transfer efficiency or may be made of a polymer material including nano carbon or a carbon tube.

In addition, the sealing film 52 also serves to quickly transfer the sample injected into the sample injection chamber 53 to the plurality of reagent storage chamber 55 through a plurality of mixing channels 54 without a force. To this end, the surface of the sealing film 52 is treated to be hydrophilic. That is, since the surface of the normal plastic sealing film 52 is hydrophobic or positively charged, it is not quickly transferred or unevenly when a sample having a charge is injected into the sample injection chamber 53. There is a problem that is distributed. In order to solve this problem, conventionally, the substrate 51 is tilted inclined or the sample is transferred using a power such as a pump or centrifugal force, but there is a problem in that the structure becomes complicated.

On the other hand, the surface of the sealing film 52 may be treated such that the surface has a hydrophilic method by chemical treatment, ultraviolet irradiation, plasma treatment or the like. Therefore, it is possible to transfer the sample in the sample injection chamber 53 to the sample injection chamber 53 with no force. By the way, the surface of the sealing film 52 may have a hydrophilicity by plasma treatment, but there is a problem that the effect of hydrophilicity easily disappears when exposed to air. In addition, the hydrophilic sealing film 52 has a problem that the sealing film 52 cannot be attached to the lower surface of the substrate 51 because the adhesive does not adhere well.

Accordingly, the micro biochip 5 of the present invention adheres the sealing film 52 to the lower surface of the substrate 51 and then performs a vacuum plasma treatment. Then, the surface of the sealing film 52 attached to the lower surface of the substrate 51 is hydrophilically treated by the plasma through the sample inlet 53a and the plurality of reagent inlets 55a of the sample inlet chamber 53. That is, only the surface of the sealing film closing the sample injection chamber 53, the plurality of reagent storage chambers 55, and the plurality of mixing channels 54 is partially hydrophilic. The hydrophilic micro biochip 5 is stored in a hermetic bag made of a thin aluminum film to be stored and distributed so that the hydrophilic effect is not maintained.

Meanwhile, a protrusion 58 is formed on the lower surface of the substrate 51 along a sample injection chamber 53, a plurality of reagent storage chambers 55, and a mixing channel 54. In addition, protrusions 59 are also formed in a quadrangular shape so as to surround them. These protrusions 58 and 59 minimize the contact area between the sealing film 52 and the substrate 51 to facilitate the adhesion of the sealing film 52. In addition, the upper surface of the substrate 51 may be provided with a sticker 57 for closing the sample inlet (53a) and a plurality of reagent inlet (55a).

On the other hand, the plurality of reagent storage chamber 55 is a reagent is injected through the reagent inlet (55a). For example, reagents include fluorescent dyes, probes, and the like. The reagent injected into the reagent storage chamber 55 may be lyophilized and adhered to the bottom of the reagent storage chamber 55, that is, the surface of the sealing film 52. At this time, the operation of injecting different reagents into the plurality of reagent storage chamber 55 can be made by an expert in the laboratory because precision is required. In the field, the sample and the reagents stored in the plurality of reagent storage chambers 55 may be mixed only by injecting the sample taken into one sample injection chamber 53.

Hereinafter, a method of quantitatively dispensing a single sample using the micro biochip according to the present invention.

4 and 5 are explanatory views showing a process of dispensing a single sample using a micro biochip according to the present invention.

First, FIGS. 4A and 5A are cross-sectional views and plan views showing an example of the micro biochip 5 according to the present invention. The micro biochip 5 of the present invention is supplied in a sealed state in an aluminum bag at the manufacturing stage. As shown, it consists of a single sample injection chamber 54, a plurality of reagent storage chamber 55 and a plurality of mixing channels 54 connecting the sample injection chamber a plurality of reagent storage chamber (55).

4B and 5B show the injection of reagents (primers and PCR MIX) into the plurality of reagent storage chambers 55 using a pipette. Preferably, the step of injecting reagents into the reagent storage chamber 55 of various types of reagents may be performed by an expert in a laboratory or using an automated device. The micro biochip 5 into which the reagent is injected is freeze-dried to fix the reagent to the bottom of the reagent storage chamber 55. In this way, fixing the reagent on the floor can prevent the reagent from moving or leaking while transferring the micro biochip 5.

4C and 5C show a state in which a sample is injected into the sample injection chamber 53. As such, the operation of injecting the sample into the sample injection chamber 53 may be performed at the site where the sample is collected. When the sample is injected into the sample injection chamber 53 by using a pipette, as shown in FIGS. 4D and 5D, the sample is immediately transferred to the plurality of reagent storage chambers 55 through the plurality of mixing channels 54. At this time, the sample is quickly transferred to the plurality of reagent storage chambers 55 without force along the hydrophilic surface of the sealing film 52. The sample transferred to the reagent storage chamber 55 is mixed with the reagent by melting the fixed reagent. The mixed solution in which the reagent and the sample are mixed is filled and mixed in the plurality of reagent storage chambers 55 and the mixing channel 54. However, the mixed liquid in the mixed channel 54 does not flow back into the sample injection chamber 53 due to the narrow channel portion 642. Finally, the sticker 57 is attached to the upper surface of the micro biochip 5 to seal the injection hole.

As described above, the sample injected into the sample injection chamber 53 is transferred to the plurality of reagent storage chambers 55 through the narrow channel portion 542 and the mixing channel portion 541. In the mixing channel part 541, the reagent supplied from the reagent storage chamber 55 and the sample supplied from the sample injection chamber 53 are mixed and reacted. On the other hand, the mixed liquid in the mixing channel part 541 does not flow back into the sample injection chamber 53 due to the narrow channel part 542. That is, since the narrow channel portion 542 has a relatively small diameter and a certain amount of sample remains inside the sample injection chamber 53, the mixed liquid in the mixing channel portion 541 does not flow back to the sample injection chamber 53. .

In addition, the present invention can transfer the sample in a non-powered manner by using the hydrophilic surface of the sealing film 52 to simplify the structure of the chip and do not use a separate power source such as a centrifuge in the field where the disease occurred Very easy to use In addition, the sample is transported through the mixed mixing channel 54 which is not stored in the sample injection chamber 53 and is sealed. Since no pipette or tip is used during the transport, foreign matter may not flow in or be mixed with other reagents. Do not. In addition, since there is no contact with the outside air, the reliability of a single sample is increased. This allows multiple diagnostics because a highly reliable single sample can be mixed with different reagents.

Although specific embodiments of the present invention have been described above, the above-described embodiments are to be considered in all respects as illustrative and not restrictive, and the scope of the present invention is defined by the appended claims rather than the detailed description. It should be construed that all changes or modifications shown and derived from the meaning and scope of the claims and equivalent concepts thereof are included in the scope of the present invention. And the terms or words used in this specification and claims are not to be construed as limiting in their usual or dictionary meanings, and the inventors will appropriately define the concept of terms in order to best describe their invention. Based on the principle that it can, it should be interpreted as meaning and concept corresponding to the technical idea of the present invention. Therefore, the embodiments described in the present specification and the configuration shown in the drawings are only the most preferred embodiments of the present invention, and do not represent all of the technical idea of the present invention, so that equivalent modifications may be substituted for them at the time of the present application. It should be understood that there may be examples.

5: micro biochip 51: substrate
52: sealing film 53: sample injection chamber
54: Mixing Channel 55: Reagent Storage Chamber
55a: reagent inlet 57: sticker
58, 59: protrusion 541: mixing channel portion
542: narrow channel portion 543: step

Claims (6)

A substrate made of a transparent material having a predetermined thickness;
A sample injection chamber formed by penetrating the substrate up and down;
A plurality of reagent storage chambers spaced apart from the sample injection chamber by a predetermined distance and formed with grooves having a predetermined depth on a lower surface of the substrate;
A mixing channel formed by forming a channel at a predetermined depth on a lower surface of the substrate so as to connect the one sample injection chamber and the plurality of reagent storage chambers;
And a sealing film attached to a lower surface of the substrate to close the open lower ends of the one sample injection chamber, the plurality of reagent storage chambers, and the plurality of mixing channels.
The sealing film has a surface that closes the open lower ends of the one sample injection chamber, the plurality of reagent storage chambers, and the plurality of mixing channels to be hydrophilic, so that the sample injected into the one sample injection chamber is connected to the plurality of mixing channels. Micro biochip for PCR, characterized in that quickly transferred to a plurality of reagent storage chamber through. According to claim 1,
Surface of the sealing film micro-biochip for PCR, characterized in that the hydrophilic treatment. The method of claim 2,
The sealing film is a micro biochip for PCR, characterized in that the surface for closing the one sample injection chamber, a plurality of reagent storage chamber and a plurality of mixing channels is hydrophilic. The method of claim 2,
The sealing film is a micro-biochip for PCR, characterized in that made of a polymeric material containing a material having excellent heat transfer rate, such as nano carbon or carbon tube. According to claim 1,
The sample injection chamber has a size such that the sample does not overflow while the injected sample is transferred to the plurality of reagent storage chambers, and a sample injection hole is formed at an upper end of the sample injection chamber, and the plurality of sample injection holes are formed on an upper surface of the substrate. A micro-biochip for PCR, characterized in that a plurality of reagent inlet is formed by forming a groove of a constant depth to be connected to the reagent storage chamber. The method of claim 5,
The mixing channel is formed by forming a groove having a predetermined width and length on a lower surface of the substrate, the groove being connected to the reagent storage chamber and extending a predetermined length toward the sample injection chamber and having a width and height similar to that of the reagent storage chamber. And a narrow channel portion connected to the sample injection chamber and extending to a predetermined length toward the reagent injection chamber and connected to the mixing channel portion, the narrow channel portion being formed with a groove having a smaller width and height than the mixing channel. PCR micro-biochip characterized in that the stepped with a constant width and height between the mixed channel portion and the narrow channel portion.

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