阅读说明：本技术 一种基于壳聚糖吸附核酸的微流控芯片及制作方法 (Microfluidic chip for adsorbing nucleic acid based on chitosan and manufacturing method ) 是由 弥胜利 陈百良 徐菲 黄嘉骏 于 2021-07-07 设计创作，主要内容包括：一种基于壳聚糖吸附核酸的微流控芯片及制作方法,该芯片包括流道层,流道层中设置有核酸提取流道,核酸提取流道为表面修饰有一层壳聚糖的PMMA流道,当向PMMA流道注入核酸溶液时,PMMA流道表面的壳聚糖在核酸溶液的第一液体环境下吸附核酸,当向PMMA流道注入洗脱液时,PMMA表面的壳聚糖在洗脱液的第二液体环境下解吸核酸,洗脱得到核酸,从而实现核酸的提取。该微流控芯片可以通过流道直接实现核酸的抓取和洗脱,不需要嵌入磁珠也不要设计微阵列,这大大降低了芯片的制造成本以及流体的控制难度,其结构简单,控制方便,能够实现快速的核酸提取,并且不会引入会对后续扩增产生影响的化学物质。(A micro-fluidic chip for adsorbing nucleic acid based on chitosan and a manufacturing method thereof are disclosed, the chip comprises a flow channel layer, a nucleic acid extraction flow channel is arranged in the flow channel layer, the nucleic acid extraction flow channel is a PMMA flow channel with a layer of chitosan modified on the surface, when nucleic acid solution is injected into the PMMA flow channel, the chitosan on the surface of the PMMA flow channel adsorbs the nucleic acid in a first liquid environment of the nucleic acid solution, when eluent is injected into the PMMA flow channel, the chitosan on the surface of the PMMA flow channel desorbs the nucleic acid in a second liquid environment of the eluent, and the nucleic acid is eluted to obtain the nucleic acid, so that the extraction of the nucleic acid is realized. This micro-fluidic chip can directly realize snatching and eluting of nucleic acid through the runner, need not imbed the magnetic bead and also need not design the microarray, this greatly reduced the manufacturing cost of chip and fluidic control degree of difficulty, its simple structure, control is convenient, can realize quick nucleic acid and draw to can not introduce the chemical substance that can produce the influence to follow-up amplification.)

1. The microfluidic chip based on the chitosan nucleic acid adsorption comprises a flow channel layer and is characterized in that a nucleic acid extraction flow channel is arranged in the flow channel layer, the nucleic acid extraction flow channel is a polymethyl methacrylate (PMMA) flow channel with a layer of chitosan modified on the surface, when a nucleic acid solution is injected into the PMMA flow channel, the chitosan on the surface of the PMMA flow channel adsorbs the nucleic acid in a first liquid environment of the nucleic acid solution, when an eluent is injected into the PMMA flow channel, the chitosan on the surface of the PMMA flow channel desorbs the nucleic acid in a second liquid environment of the eluent, and the nucleic acid is obtained through elution, so that the extraction of the nucleic acid is realized.

2. The microfluidic chip according to claim 1, wherein the chitosan is modified to the PMMA flow channel by uv irradiation through EDC-NHS cross-linking reaction.

3. The microfluidic chip according to claim 2, wherein the chitosan is immobilized on the surface of the flow channel by immersing the PMMA flow channel irradiated with uv in a mixed solution of EDC-NHS and chitosan in MES buffer, EDC reacts with carboxyl groups on the surface of PMMA to generate intermediate ester, and the intermediate stabilized by NHS reacts with amino groups of chitosan.

4. The microfluidic chip according to any of claims 1 to 3, wherein the microfluidic chip is a finger-pressure type driving structure, the finger-pressing type driving structure comprises a liquid inlet, a finger-pressing cavity, a vent for communicating the finger-pressing cavity with the atmosphere, a valve with a weir structure, a liquid storage cavity, the nucleic acid extraction flow channel and a liquid outlet, wherein the liquid inlet, the valve and the liquid storage chamber are connected in sequence and provided with three groups, each group is respectively used for injecting nucleic acid solution, washing solution and eluent, and each group is connected to the inlet of the nucleic acid extraction flow channel, the outlet of the nucleic acid extraction flow channel is connected with the liquid outlet, the finger pressure chamber in each group is connected with the valve, the air pressure difference change caused by pressing the finger pressure chamber by a finger is utilized to control the flow of liquid and the opening and closing of the valve, and the liquid storage chamber is used for temporarily storing the liquid injected from the liquid inlet.

5. The microfluidic chip according to claim 4, wherein the microfluidic chip has a four-layer structure including a cover plate, an air channel layer, a thin film layer and the flow channel layer in sequence from top to bottom, the finger pressure chamber is formed on the air channel layer, the valve is formed on the air channel layer, the thin film layer and the flow channel layer, and the thin film layer deforms under the action of air pressure difference to open or close the valve.

6. The microfluidic chip according to claim 5, wherein the cover plate, the gas channel layer, and the thin film layer are made of Polydimethylsiloxane (PDMS) elastic material.

7. The microfluidic chip of claim 5, wherein the four layers are sealed using a bolt-on seal.

8. The method for manufacturing the microfluidic chip based on nucleic acid adsorption of chitosan according to any one of claims 1 to 7, comprising the following steps:

molding a runner layer with a PMMA runner;

irradiating the flow channel layer with ultraviolet rays;

and soaking the PMMA flow channel in a mixed solution of EDC-NHS and chitosan, wherein mes buffer solution is used as a solvent, EDC reacts with carboxyl on the surface of PMMA to generate intermediate ester, the intermediate is kept stable by NHS, and the intermediate reacts with amino of chitosan to fix the chitosan on the surface of the flow channel.

9. The method of claim 8, further comprising:

and forming a cover plate, an air channel layer and a thin film layer of the PDMS material, baking, performing oxygen plasma, incubating and sealing, and then fastening the cover plate, the air channel layer and the thin film layer with the air channel layer through bolts.

10. A nucleic acid extraction method using the chitosan nucleic acid adsorption-based microfluidic chip of any one of claims 1 to 7.

Technical Field

The invention relates to a nucleic acid extraction device, in particular to a micro-fluidic chip for adsorbing nucleic acid based on chitosan and a preparation method thereof.

Background

The rapid and reliable pathogen detection has important significance in the fields of food safety, water quality, clinical analysis, bioterrorism defense and the like. Traditional microbiological methods require the cultivation of cells or bacteria, are time consuming and inefficient, and are only suitable for organisms that can be grown under laboratory conditions. Thus, the Polymerase Chain Reaction (PCR) technique involving real-time detection is a more widely used technique today. However, many of the obtained samples (e.g., whole blood, urine, etc.) have low nucleic acid concentrations and contain many impurities (e.g., proteins, inorganic salts, etc.) that interfere with amplification, and direct amplification does not yield desirable results. Most samples therefore require pretreatment and nucleic acid extraction prior to amplification. The commonly used nucleic acid extraction kit usually depends on laboratory conditions, has a complex operation process, is not suitable for field diagnosis, has poor air tightness, is easy to generate cross contamination and the like.

The microfluidic chip is used as a novel research tool, plays a crucial role in biochemical analysis in a laboratory, and is developed to be used for field detection in various scenes. Compared with the traditional laboratory detection method, the micro-fluidic chip-based detection method has the advantages that the sample size is small, the manual operation is less, and the detection time and the sample consumption in the process are greatly reduced.

It is to be noted that the information disclosed in the above background section is only for understanding the background of the present application and thus may include information that does not constitute prior art known to a person of ordinary skill in the art.

Disclosure of Invention

The invention mainly aims to overcome the defects of the background technology and provide a micro-fluidic chip for adsorbing nucleic acid based on chitosan and a manufacturing method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

the microfluidic chip comprises a flow channel layer, wherein a nucleic acid extraction flow channel is arranged in the flow channel layer, the nucleic acid extraction flow channel is a polymethyl methacrylate (PMMA) flow channel with a layer of chitosan modified on the surface, when a nucleic acid solution is injected into the PMMA flow channel, the chitosan on the surface of the PMMA flow channel adsorbs nucleic acid in a first liquid environment of the nucleic acid solution, when an eluent is injected into the PMMA flow channel, the chitosan on the surface of the PMMA flow channel desorbs the nucleic acid in a second liquid environment of the eluent, and the nucleic acid is obtained by elution, so that the extraction of the nucleic acid is realized.

Further:

and the chitosan is modified on the PMMA flow channel irradiated by ultraviolet through EDC-NHS crosslinking reaction.

Soaking a PMMA flow channel subjected to ultraviolet irradiation in a mixed solution of EDC-NHS and chitosan, which takes MES buffer solution as a solvent, reacting EDC with carboxyl on the surface of PMMA to generate intermediate ester, and reacting the intermediate, which is kept stable by NHS, with amino of chitosan so as to fix the chitosan on the surface of the flow channel.

Micro-fluidic chip is finger-pressure formula drive structure, finger-pressure formula drive structure includes inlet, finger-pressure cavity, intercommunication finger-pressure cavity and atmospheric blow vent, have valve, liquid storage cavity of weir structure nucleic acid draws runner and liquid outlet, wherein the inlet the valve liquid storage cavity connects gradually and is provided with three groups, and every group is used for injecting into nucleic acid solution, washing liquid and eluant respectively, and every group all is connected to the entry of nucleic acid extraction runner, nucleic acid draws the exit linkage of runner the liquid outlet, in every group finger-pressure cavity with the valve links to each other, utilizes the finger to press the poor flow of change control liquid of atmospheric pressure that finger-pressure cavity caused with the opening and shutting of valve, liquid storage cavity is used for temporary storage follow the liquid that the inlet was injected into.

The micro-fluidic chip is from last to including apron, air flue layer, thin layer down in proper order and the four-layer structure on flow channel layer, the finger pressure cavity forms on the air flue layer, the valve forms the air flue layer the thin layer with on the flow channel layer, the thin layer warp in order to realize opening or closing of valve under the effect of atmospheric pressure difference.

The cover plate, the air channel layer and the thin film layer are all made of Polydimethylsiloxane (PDMS) elastic materials.

And the four layers are fastened and sealed by using bolts.

A manufacturing method of the microfluidic chip based on nucleic acid adsorption of chitosan comprises the following steps:

molding a runner layer with a PMMA runner;

irradiating the flow channel layer with ultraviolet rays;

and soaking the PMMA flow channel in a mixed solution of EDC-NHS and chitosan, wherein MES buffer solution is used as a solvent, EDC reacts with carboxyl on the surface of PMMA to generate intermediate ester, the intermediate is kept stable by NHS, and the intermediate reacts with amino of chitosan to fix the chitosan on the surface of the flow channel.

Also comprises the following steps:

and forming a cover plate, an air channel layer and a thin film layer of the PDMS material, baking, performing oxygen plasma, incubating and sealing, and then fastening the cover plate, the air channel layer and the thin film layer with the air channel layer through bolts.

A nucleic acid extraction method using the microfluidic chip based on chitosan nucleic acid adsorption.

The invention has the following beneficial effects:

the invention provides a micro-fluidic chip for nucleic acid extraction, wherein a nucleic acid extraction flow channel is arranged in a flow channel layer, the nucleic acid extraction flow channel is made of PMMA material, a layer of chitosan is modified on the surface of the PMMA flow channel, when a nucleic acid solution is injected into the PMMA flow channel, the chitosan on the surface of the PMMA flow channel adsorbs nucleic acid in a first liquid environment of the nucleic acid solution, when an eluent is injected into the PMMA flow channel, the chitosan on the surface of the PMMA flow channel desorbs the nucleic acid in a second liquid environment of the eluent, and the nucleic acid is eluted, so that the nucleic acid is extracted. The extraction principle utilizes the mechanism that chitosan can adsorb nucleic acid under an acidic environment. The micro-fluidic chip can directly realize the grabbing and elution of nucleic acid through the flow channel without embedding magnetic beads or designing a micro-array, thereby greatly reducing the manufacturing cost of the chip and the control difficulty of fluid. The cost of the chip is lower than that of the existing commercial kit, and the chip has similar extraction effect with the existing commercial kit, and can be widely applied to the field of rapid diagnosis of nucleic acid molecules.

Drawings

FIG. 1 is a schematic structural diagram of a microfluidic chip for nucleic acid extraction based on chitosan adsorption according to an embodiment of the present invention;

FIG. 2 is a schematic view of the integrated flow channel of FIG. 1;

FIG. 3 is a schematic perspective view of the microfluidic chip shown in FIG. 1;

FIG. 4 is a schematic flow chart of PMMA-modified chitosan according to an embodiment of the present invention.

Detailed Description

The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.

It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly on the other element or be indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or be indirectly connected to the other element. In addition, the connection may be for either a fixed or coupled or communicating function.

It is to be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in an orientation or positional relationship indicated in the drawings for convenience in describing the embodiments of the present invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed in a particular orientation, and be in any way limiting of the present invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the embodiments of the present invention, "a plurality" means two or more unless specifically limited otherwise.

Referring to fig. 1 to 3, an embodiment of the present invention provides a microfluidic chip for adsorbing nucleic acid based on chitosan, including a channel layer 4, a nucleic acid extraction channel 10 is disposed in the channel layer 4, the channel 10 is a PMMA channel modified with a layer of chitosan, which is made of polymethyl methacrylate (PMMA) material, when a nucleic acid solution is injected into the channel 10, the chitosan on the surface of the channel 10 adsorbs nucleic acid in a first liquid environment of the nucleic acid solution, and when an eluent is injected into the channel 10, the chitosan on the surface of the channel 10 desorbs nucleic acid in a second liquid environment of the eluent, and the nucleic acid is eluted, so as to achieve extraction of nucleic acid.

According to the chip disclosed by the invention, the chitosan with the functional group capable of extracting nucleic acid is integrated on the flow channel of the chip, the nucleic acid is extracted by directly utilizing the flow channel, magnetic beads do not need to be embedded, and a nucleic acid extraction chamber with a microarray does not need to be designed, so that the manufacturing cost of the chip and the control difficulty of fluid are greatly reduced. The mechanism of the chip for extracting nucleic acid is based on the difference of the adsorption of chitosan to nucleic acid under the environment with different pH values and salt ion concentrations, in the whole extraction process, the adsorption and desorption of nucleic acid can be completed only by properly changing the pH value and the salt ion concentration in the liquid environment, and the eluent does not contain substances which can influence the subsequent amplification step, such as ethanol and the like.

In a preferred embodiment, the chitosan is modified onto the PMMA flow channel irradiated with uv light by EDC-NHS cross-linking reaction.

In a preferred embodiment, the PMMA flow channel irradiated by ultraviolet is soaked in EDC-NHS and chitosan mixed solution which takes MES buffer solution as solvent, EDC reacts with carboxyl on the surface of PMMA to generate intermediate ester, and the intermediate which is kept stable by NHS reacts with amino of chitosan to fix the chitosan on the surface of the flow channel.

Referring to fig. 1 to 3, in a preferred embodiment, the microfluidic chip is a finger-pressing type driving structure, the finger-pressing type driving structure includes a liquid inlet 5, a finger-pressing chamber 7, a vent 8 communicating the finger-pressing chamber 7 with the atmosphere, a valve 6 with a weir structure, a liquid storage chamber 9, a nucleic acid extraction flow channel 10, and a liquid outlet 11, wherein the liquid inlet 5, the valve 6, and the liquid storage chamber 9 are sequentially connected and provided with three sets, each set is used for injecting a nucleic acid solution, a washing solution, and an eluent, each set is connected to an inlet of the nucleic acid extraction flow channel 10, an outlet of the nucleic acid extraction flow channel 10 is connected to the liquid outlet 11, the finger-pressing chamber 7 in each set is connected to the valve 6, and the air pressure difference change caused by pressing the finger-pressing chamber 7 controls the flow of liquid and the opening and closing of the valve 6, the liquid storage chamber 9 serves to temporarily store the liquid injected from the loading port 5. Through adopting the finger-pressing type driving structure, the microfluid is controlled only by pressing the finger as the driving force without any exogenous supporting equipment, thereby not only facilitating the use, but also saving the related equipment and consumables.

Referring to fig. 1 and 3, in a preferred embodiment, the microfluidic chip includes a four-layer structure including a cover plate 1, an air channel layer 2, a thin film layer 3, and the flow channel layer 4 in sequence from top to bottom, the finger pressure chamber 7 is formed on the air channel layer 2, the valve 6 is formed on the air channel layer 2, the thin film layer 3, and the flow channel layer 4, and the thin film layer deforms under the action of an air pressure difference to open or close the valve 6. The specific implementation of the valve 6 can adopt (but is not limited to) the structure disclosed in the prior application CN 112195099A.

In a preferred embodiment, the cover plate 1, the air channel layer 2 and the thin film layer 3 are made of Polydimethylsiloxane (PDMS) elastic material.

In a preferred embodiment, the four layers are sealed using bolt fastening.

The embodiment of the invention also provides a manufacturing method of the micro-fluidic chip based on nucleic acid adsorption by chitosan, which comprises the following steps:

molding a runner layer with a PMMA runner;

irradiating the flow channel layer with ultraviolet rays;

and soaking the PMMA flow channel in a mixed solution of EDC-NHS and chitosan, wherein MES buffer solution is used as a solvent, EDC reacts with carboxyl on the surface of PMMA to generate intermediate ester, the intermediate is kept stable by NHS, and the intermediate reacts with amino of chitosan to fix the chitosan on the surface of the flow channel.

In a preferred embodiment, the method further comprises the steps of:

and forming a cover plate, an air channel layer and a thin film layer of the PDMS material, baking, performing oxygen plasma, incubating and sealing, and then fastening the cover plate, the air channel layer and the thin film layer with the air channel layer through bolts.

The embodiment of the invention also provides a nucleic acid extraction method using the chitosan-based nucleic acid adsorption microfluidic chip of any one of the preceding embodiments.

In some embodiments, a microfluidic chip for nucleic acid extraction using finger pressing as a driving force is integrally divided into a cover plate, an air channel layer, a thin film layer and a flow channel layer, and the structure of the microfluidic chip comprises a liquid inlet, a finger pressing chamber, a valve module with a weir structure, a liquid storage chamber, a flow channel for nucleic acid extraction, a waste liquid collecting pipeline and the like. The finger pressure chamber utilizes the air pressure difference caused by finger pressing so as to control the flow of liquid and the opening and closing of the valve; the liquid storage chamber is used for temporarily storing the liquid injected from the liquid inlet so as to be convenient for controlling the flow of the liquid in the chip; the flow channel for nucleic acid extraction adopts a polymethyl methacrylate (PMMA) material modified with chitosan, and the extraction principle utilizes the mechanism that chitosan can adsorb nucleic acid in an acidic environment. The three-layer structure of the cover plate, the air channel layer and the film layer is made of Polydimethylsiloxane (PDMS) elastic materials. The four layers are fastened and sealed by bolts.

In some embodiments, the chip is provided with three liquid inlets, which correspond to the nucleic acid stock solution to be extracted, the washing solution and the eluent, respectively, and the three liquids flow into the liquid storage chamber after being injected. The nucleic acid extraction flow channel has a large surface area, the surface of the PMMA has a layer of chitosan, and after liquid in the liquid storage chamber enters the flow channel and is fully mixed, nucleic acid is combined with the chitosan on the surface, so that the effect of extracting nucleic acid is achieved.

In some embodiments, the injected liquid may flow into the liquid storage chamber when the finger pressure chamber is not depressed. When the finger pressure chamber is pressed, the valve on the left side is closed, the valve on the right side is opened, and the stored liquid flows into the next flow passage. The liquid can not directly pass through the valve, and when the finger pressing chamber is pressed down or loosened, the generated air pressure difference can adjust the opening and closing states of the two valves.

In some embodiments, a microfluidic chip for nucleic acid extraction, the step of nucleic acid extraction comprising:

(1) injecting nucleic acid stock solution to be extracted, washing solution and eluent from three liquid inlets respectively;

(2) pressing a finger pressure chamber beside the nucleic acid stock solution chamber, injecting the nucleic acid stock solution into the nucleic acid extraction flow channel, and waiting for a certain time (5-10 minutes) to ensure that the solution is fully contacted with the flow channel to complete the grabbing of the nucleic acid;

(3) pressing a finger pressing chamber beside the washing liquid chamber, injecting the washing liquid into the nucleic acid extraction flow channel, and continuing pressing after waiting for a certain time to enable the washing liquid to flow into a liquid outlet from the nucleic acid extraction flow channel so as to wash away residual nucleic acid stock solution;

(4) pressing a finger pressing cavity beside the eluent cavity, injecting the eluent into the nucleic acid extraction flow channel, and continuing to press after waiting for a certain time (5-10 minutes) so that the eluent flows into a liquid outlet from the nucleic acid extraction flow channel to obtain the nucleic acid obtained by elution;

specific embodiments of the present invention are described further below with reference to the accompanying drawings.

A finger-pressure portable-driven micro-fluidic chip for nucleic acid extraction and purification and real-time fluorescence nucleic acid amplification detection is shown in figures 1 and 3 and is a structural schematic diagram of the nucleic acid detection chip, and the chip comprises a cover plate (1), an air channel layer (2), a thin film layer (3) and a flow channel layer (4).

As shown in FIG. 2, the whole chip comprises a liquid inlet (5), a valve (6) with a weir structure, a finger pressure chamber (7), a vent (8) for communicating the finger pressure chamber with the atmosphere, a liquid storage chamber (9), a flow channel (10) for nucleic acid extraction, and a liquid outlet (11).

As shown in FIG. 4, the chitosan was modified onto the UV-irradiated PMMA flow channel using EDC-NHS cross-linking reaction.

The chip is made of three PDMS layers and one PMMA layer and comprises a cover plate, an air channel layer, a film layer and a flow channel layer. The mold for the PDMS layer was fabricated by photolithography. Subsequently, SU-82100 was spin coated to obtain a thickness of 300 μm. In the first step, a photoresist is spin-coated, then exposed to ultraviolet light, and then washed by a developing solution to obtain a corresponding structure. By mixing PDMS precursor with curing agent in a 10: 1 and cured at 80 c for 2 hours to obtain an airway layer. Thereafter, the reservoir was formed with a through hole having a diameter of 4.0 mm. The covering layer is prepared by mixing PDMS and a curing agent in a ratio of 10: 1 onto a bare silicon wafer and cured at 150 c for 5 minutes. Then, a flat needle (19G) is used to perforate the entrance. The flow channel layer is processed and manufactured by PMMA, after processing, ultraviolet irradiation is carried out for 30 minutes, and then the flow channel layer is soaked in a mixed solution of EDC-NHS and chitosan which takes MES buffer solution as a solvent, EDC reacts with carboxyl on the surface of PMMA to generate intermediate ester, NHS enables the intermediate ester to keep stable, and the intermediate reacts with amino of chitosan and fixes the chitosan on the surface of the flow channel. Subsequently, the PDMS was baked at 80 ℃ for 2 h. The three layers were treated with oxygen plasma for 120 seconds and incubated at 65 ℃ for 10 minutes to seal the three layers before being bolted to the PMMA layer.

The nucleic acid detection step specifically comprises:

(1) injecting 40 mu L of nucleic acid stock solution to be extracted from the liquid inlet (5) by using an injection gun, pressing the finger pressure chamber (7) to enable the finger pressure chamber to enter the liquid storage chamber (9) and the flow channel (10) for extracting the nucleic acid, and waiting for 5-10 minutes for the nucleic acid to be fully combined with the flow channel;

(2) injecting 40 mu L of washing solution from the liquid inlet (5) by using an injection gun, pressing the finger pressure chamber (7) into the liquid storage chamber (9) and the flow channel (10) for extracting nucleic acid, and repeating the steps for three times;

(3) injecting 40 mu L of eluent from the liquid inlet (5) by using an injection gun, pressing the finger pressure chamber (7) to enable the finger pressure chamber to enter the liquid storage chamber (9) and the flow channel (10) for extracting nucleic acid, waiting for 5-10 minutes for the eluent to fully contact with the flow channel, then obtaining the eluent from the liquid outlet (11) for subsequent detection or amplification.

The background of the present invention may contain background information related to the problem or environment of the present invention and does not necessarily describe the prior art. Accordingly, the inclusion in the background section is not an admission of prior art by the applicant.

The foregoing is a more detailed description of the invention in connection with specific/preferred embodiments and is not intended to limit the practice of the invention to those descriptions. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the described embodiments without departing from the spirit of the invention, and these substitutions and modifications should be considered to fall within the scope of the invention. In the description herein, references to the description of the term "one embodiment," "some embodiments," "preferred embodiments," "an example," "a specific example," or "some examples" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Various embodiments or examples and features of various embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction. Although embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the scope of the claims.