KR102053404B1 - Pretreatment system for concentration and purification of samples - Google Patents

Abstract

The present invention relates to a pretreatment system for concentrating and purifying a sample, more specifically a sample storage cartridge; A pinch-solenoid valve system connected to the sample storage cartridge; A lower module connected to the valve system; An upper module positioned above the lower module; A microfluidic chip mounted on the lower module; And a tubing interlocking pump connected to the microfluidic chip, to provide a pretreatment system for concentrating and purifying a sample. Accordingly, the present invention provides a low concentration pathogen sample, which is difficult to detect by nucleic acid amplification techniques, to a detectable concentration through pretreatment concentration, removes molecular diagnostic inhibitors in the sample, and purifies nucleic acid from the concentrated pathogen. The post-extraction process is fully automated and can be easily used by non-skilled individuals to provide a pretreatment system with increased convenience and reproducibility.

Description

Pretreatment System for Concentration and Purification of Samples {PRETREATMENT SYSTEM FOR CONCENTRATION AND PURIFICATION OF SAMPLES}

The present invention relates to a pretreatment system for the concentration and purification of a sample.

In the present hospital, in order to detect low concentrations of pathogens by molecular diagnostic techniques such as nucleic acid amplification, the pretreatment process is incubated for more than 12 hours. After that, the cultured sample is used by concentrating the pathogen through a centrifuge. This method takes a long time and there is a molecular diagnostic inhibitor in the sample to be detected, which affects the sensitivity of the molecular diagnostic technique. In addition, this pretreatment process is performed by the researchers and is affected by the proficiency of the researchers, and may cause the infection problem of the researchers due to exposure to high-risk pathogens.

In the case of pathogen enrichment technology, recently, Immunomagnetic separation (IMS), a method of binding an antibody to the surface of magnetic nanoparticles and reacting with a specific bacterium, can be replaced. The collection efficiency of the particles is reduced, and it takes a lot of time due to the complicated experiment process.

Nucleic acid purification methods generally have a technique using a centrifuge. This process has a disadvantage in that the centrifugation is performed while changing the buffer solution repeatedly, such as the process of concentrating bacteria and washing process using a centrifuge. There is a purification technology using magnetic silica particles as an alternative technique, but this technique also consists of a complex method that must proceed while repeatedly changing the buffer solution in the tube.

Molecular diagnostic starter companies such as Qiagen, Cephied, abbott, and BD have developed commercial kits for extracting genes directly from pathogens in a sample.

On the other hand, as a study to rapidly deliver a chemical sample, such as a microfluidic channel, or a liquid sample such as blood or biomolecules in the desired direction (Korean Patent Publication No. 2007-0005153), these channels are Even with very few samples and samples, chemical detection, reaction testing, drug development, cell culture, biological organ simulations, or experimentation can be performed in intensively designed chips. Recently, research on open fluidic channels, which minimize the amount of sample required for analysis and analyzes with a single drop of liquid, is also underway. In such a micro fluidic channel, it is important to control the flow of the fluid, for example, to control the flow of the fluid using electricity or a three-dimensional structure.

The present inventors earnestly researched to develop a microfluidic chip and a system for automating a sample pretreatment process capable of rapidly concentrating low concentrations of pathogens present in a sample and simultaneously purifying and extracting pure nucleic acids from the concentrated pathogens. The present invention was completed by developing a pretreatment system through assembly and automatic control of a sample storage cartridge, a pump, a valve, a heater, a permanent magnet, and a vibration motor.

Accordingly, it is an object of the present invention to provide a pretreatment system for concentration and purification of a sample.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention comprises a sample storage cartridge; A pinch-solenoid valve system connected to the sample storage cartridge; A lower module connected to the valve system; An upper module positioned above the lower module; A microfluidic chip mounted on the lower module; And a tubing interlocking pump connected to the microfluidic chip, to provide a pretreatment system for concentrating and purifying a sample.

In one embodiment of the present invention, the sample storage cartridge is characterized by storing a sample, a lysis buffer, an elution buffer and a wash buffer.

In another embodiment of the present invention, the lower module is characterized in that the permanent magnet and the electric motor is provided.

In another embodiment of the present invention, the upper module is characterized in that the film valve actuator for operating the film valve of the heater and the microfluidic chip.

In another embodiment of the invention, the microfluidic chip is a substrate; A sample injection hole formed on the substrate; A channel forming a movement path of the sample; A purification chamber connected to the channel; An impurity removal outlet through which impurities removed from the purification chamber are discharged; An air inlet through which air is injected into the channel; And a purified nucleic acid outlet through which the purified sample exits the purification chamber.

As another embodiment of the present invention, the sock end of the purification chamber is characterized in that the film valve is provided.

As another embodiment of the present invention, the lower portion of the purification chamber is characterized in that the permanent magnet is provided.

In another embodiment of the present invention, the microfluidic chip further comprises a vibration motor.

In the pretreatment system using a microfluidic chip according to the present invention, a pretreatment process (bacterial concentration, impurity separation, nucleic acid purification and extraction) of a sample for separating pure nucleic acid to be used in molecular diagnostic techniques can be performed within 1 hour, Each process can be performed independently in a single microfluidic chip, and since the concentration and nucleic acid purification are simultaneously performed in one reaction space, the sample loss is small and thus it is sensitively excellent.

In addition, to automate the sample pretreatment process in the microfluidic chip, an automated system consisting of module assembly such as tubing pump, pinch valve, heater, and permanent magnet was developed so that it can be easily used by unskilled people, thereby increasing convenience and reproducibility.

1 shows a view of a microfluidic chip of the present invention.
Figure 2 is a view showing the microfluidic chip and each component manufactured in the present invention.
3 is an exploded view of the microfluidic chip of the present invention.
Figure 4a is a view showing the upper and lower frames and the main substrate of the microfluidic chip of the present invention.
Figure 4b is a view showing the coupling of the lower frame and the main substrate of the microfluidic chip of the present invention.
Figure 4c shows a picture of assembling the microfluidic chip of the present invention.
5A is a diagram illustrating a configuration of an automated sample preparation system of the present invention.
5B shows the microfluidic chip of the present invention mounted to the device stage of the system.
Figure 6a is a view showing a system manufactured in an embodiment of the present invention, including an upper module and a lower module.
Figure 6b is a view showing the internal structure of the system produced in the embodiment of the present invention.
Figure 6c is a view showing in more detail the upper module and the lower module of the system manufactured in the embodiment of the present invention.
7A is a view showing the top of the system fabricated in the embodiment of the present invention.
7B is a view showing the right side of the system manufactured in the embodiment of the present invention.
8 is a schematic view of a pretreatment automation system for concentration and purification of a sample of the present invention.
9 is a view showing a process of the pretreatment method for the concentration and purification of the sample of the present invention.
10 is a view showing the results of gel electrophoresis of amplified products after PCR (nucleic acid amplification) is performed on the raw material and the sample subjected to the pretreatment method of the present invention without the pretreatment method of the present invention.
11 is a view showing the results of fluorescence intensity after performing a quantitative nucleic acid amplification technique (quantitative PCR) on the raw material and the sample subjected to the pretreatment method of the present invention without the pretreatment method of the present invention.

The present inventors make a low concentration pathogen sample, which is difficult to detect by nucleic acid amplification techniques, to a detectable concentration through concentration of pretreatment, removes molecular diagnostic inhibitors in the sample, and extracts nucleic acid after purification from the concentrated pathogen. As a result of research to develop automated pretreatment system that can concentrate and purify microfluidic chip and sample which can be processed simultaneously, the pretreatment through assembly and automatic control of sample storage cartridge, pump, valve, heater, permanent magnet and vibration motor When using the system, the present invention has been completed by confirming that the concentration and purification of samples capable of molecular diagnostics can be performed by an automated system.

Thus, the present invention, a sample storage cartridge; A pinch-solenoid valve system connected to the sample storage cartridge; A lower module connected to the valve system; An upper module positioned above the lower module; A microfluidic chip mounted on the lower module; And a tubing interlocking pump connected to the microfluidic chip, to provide a pretreatment system for concentrating and purifying a sample.

The present invention also provides a substrate; A sample injection hole formed on the substrate; A channel forming a movement path of the sample; A purification chamber connected to the channel; An impurity removal outlet through which impurities removed from the purification chamber are discharged; An air inlet through which air is injected into the channel; And a purified sample outlet from which the purified sample exits the purification chamber.

In addition, in one embodiment of the present invention was carried out a pre-treatment for the concentration and purification of the sample through a system comprising the microfluidic chip, the present invention provides a pretreatment method for the concentration and purification of the sample comprising the following steps Can provide:

Preparing a sample by mixing magnetic nanoparticles having antigens bound to a pathogen and a surface (S1);

Injecting the sample of the step S1 into the microfluidic chip filled with magnetic particles through an injection hole to concentrate the sample, and then removing the remaining fluid through the impurity removal outlet (S2);

Removing the remaining fluid in step S2, injecting lysis and binding buffers to vortex and injecting wash buffer to remove the remaining fluid through the impurity removal outlet (S3);

Removing the remaining fluid in step S3, injecting an elution buffer, and heating and vortexing the chamber to 50 to 80 ° C. (S4); And

When the vortexing of the step S4 is completed, blocking the sample inlet and the impurity removal outlet, and extracting the concentrated and purified sample by opening the air inlet and the purified nucleic acid outlet (S5).

Hereinafter, the present invention will be described in more detail with reference to the drawings.

[Fluid Chip]

The microfluidic chip of the present invention comprises a substrate as described above; A sample injection hole formed on the substrate; A channel forming a movement path of the sample; A purification chamber connected to the channel; An impurity removal outlet through which impurities removed from the purification chamber are discharged; An air inlet through which air is injected into the channel; And a purified sample outlet through which the purified sample exits the purification chamber, and is a microfluidic chip used for concentrating and purifying the sample. A diagram of the microfluidic chip of the present invention is shown in FIG.

As can be seen from the bottom of Figure 2, the present invention is an alignment hole for fixing to the substrate, a groove for the vibration motor contact can be formed, the microfluidic chip including the substrate is to use a plastic film material, As shown in Fig. 3, a thin plastic film of polyethylene terephthalate (PET), polyvinyl chloride (PVC), and polyimide (PI) is cut to form a laminated structure by cutting required shapes.

The substrate may be completed by assembling the upper and lower case of the acrylic material. The microfluidic chip of the present invention transmits four microchannels, one pathogen enrichment-nucleic acid purification chamber, four inlets and two channel valves, six hole sections for fixing the system stage, and vibration for mixer and bacterial dissolution. It consists of one hole structure, which is the contact part with the vibration motor for the motor, and eight small holes for case assembly. Each of the two inlets and two outlets consists of a sample inlet, an air inlet, an impurity outlet, and a purified nucleic acid outlet, each microchannel may be 0.5-3 mm wide and 50-200 μm high, and the concentrated-nucleic acid purification chamber is Large central rectangle (500-1000 μm high, 5-20 mm long, 1-10 mm wide) and trapezoidal shape (500-1000 μm high, 0.1-2 mm short, 2.5-5 mm long) 1 to 3 mm in length between the two sides) and the rectangle may be combined and the total allowable sample volume may be 20 to 100 μL in volume.

Film valves are provided at both ends of the purification chamber. The film valve is provided to control that the sample entering the chamber is introduced or discharged out through the channel, the film valve is to be locked in the process of the concentration and purification of the sample entering the chamber.

The lower portion of the purification chamber is provided with a permanent magnet, the lower portion of the purification chamber is provided with a permanent magnet between the magnetic nanoparticles and the permanent magnet when the sample containing a pathogen bound to the antibody of the magnetic nanoparticles Due to its magnetism, efficient concentration can be achieved.

The microfluidic chip may be filled with magnetic silica particles through an injection hole before injection of a sample, and may include a vibration motor to help mix the sample.

[sample]

The sample used in the present invention includes a pathogen and magnetic nanoparticles having an antibody bound to the surface thereof. After the antibody is bound to the surface of the magnetic nanoparticles to synthesize immunomagnetic nanoparticles, the pathogen is mixed. To bind to the antibody of the immunomagnetic nanoparticles.

The pathogen may be a virus, bacteria, fungi, or the like, and may be Escherichia coli as in the embodiment of the present invention, but is not limited thereto.

[Sample pre-processing automation system]

The microfluidic chip of the present invention is mounted on an automated sample pretreatment system to perform concentration and purification of a sample. The system of the present invention includes a sample storage cartridge; A pinch-solenoid valve system connected to the sample storage cartridge; A lower module connected to the valve system; An upper module positioned above the lower module; A microfluidic chip mounted on the lower module; And a tubing interlocking pump connected to the microfluidic chip.

Sample storage cartridge

The sample storage cartridge of the present invention is a sample storage portion in the form of a cartridge in which a sample, a lysis buffer, an elution buffer, a washing buffer, and the like may be stored. Detachable form.

The sample storage cartridge is connected to a pinch-solenoid valve system.

-Pinch-Solenoid Valve System

The pinch-solenoid valve system connected to the sample storage cartridge can control the flow of the fluid sample by the solenoid plunge press, and can be connected to the lower module on which the microfluidic chip is mounted.

-Lower module

The lower module is connected to the valve system, and is provided with a permanent magnet and an electric motor, and may serve to fix the microfluidic chip to the system and to concentrate and mix a sample of the chamber in the microfluidic chip.

The single-axis drive of the permanent magnet can automate the concentration and purification technology.

Upper module

The upper module is positioned above the lower module, and is provided with a heater so that nucleic acids can be separated from the magnetic silica particles injected into the microfluidic chip mounted on the lower module. Both ends of the heater are provided with a film valve actuator that can control the opening and closing of the film valve.

-Tubing linked pump

In the present invention, the microfluidic chip mounted on the lower model is provided with a tubing interlocking pump, and the pump may enable automatic transport of fluid samples, buffers, and the like. The tubing interlocking pump is capable of controlling the flow of fluid at 0.1 to 2.5 mL / min.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example 1 Preparation of Concentrated-Refined Microfluidic Chips

Concentrated-purified microfluidic chips were prepared according to FIG. 3. As shown in FIG. 3, after the polyimide channel forming substrate was laminated between the lower plate and the upper plate of the polyethylene material, an inlet and an outlet part of the PVC material were formed, and a film valve was formed on the sock end of the chamber. Chamber-valve) and chamber lids were stacked to make the main device portion. The main device was then housed in the upper and lower frames.

As shown in FIG. 4A, the upper frame and the lower frame have a tubing connection portion connected to the tubing pump of the sample storage cartridge, and the lower frame has a recessed recess to fix the portion of the purification chamber. The upper and lower frames were formed with alignment holes and vibration motor connecting holes that can be fixed to the nucleic acid purification system.

Thereafter, as shown in FIG. 4B, the main device was laminated to the lower frame, and then the upper frame was laminated and assembled. The assembled microfluidic chip is as shown in Figure 4c.

In Example 1, a thin plastic film made of polyethylene terephthalate (PET), polyvinyl chloride (PVC), and polyimide (PI) was cut to form a laminated structure, and an upper and lower case made of acrylic was assembled. Was used. The microfluidic chip has four microchannels, one pathogen enrichment-nucleic acid purification chamber, four inlets and two channel valves, six hole sections for fixing the system stage, a mixer and vibrations for transmitting vibrations for bacterial dissolution. It consists of a one-hole structure that is in contact with the motor and eight small holes for case assembly. The two inlets and two outlets consist of a sample inlet, an air inlet, an impurity outlet, and a purified nucleic acid outlet. Each microchannel is 1 mm wide and 100 μm high, and the enrichment-nucleic acid purification chamber has a large rectangular center (height). 800 μm, length, 13.3 mm, width 3 mm), a rectangular trapezoidal shape (800 μm in height, short 1 mm in length, 3 mm in length, 2 mm in length between two sides) and a rectangle with a volume of 40 μL It is designed and manufactured as.

Example 2 Configuration of Automated Sample Preparation System

In this example, the sample pretreatment automation system to which the microfluidic chip manufactured in Example 1 is connected was manufactured. The configuration of the system used in the present invention is shown in FIG. 5, which is composed of a sample storage cartridge, a pinch-solenoid valve system, a device stage and a heater, and a tubing interlocking pump in which a sample is largely stored as shown in FIG. 5A. As shown in FIG. 5B, the microfluidic chip was mounted on the device stage and fixed. An external photograph of the casing of the system is shown in FIG. 6A. As shown in FIG. 6A, the upper module and the lower module are included. The upper module includes a device stage and a heater, and the lower module is manufactured to include a vibration motor and a permanent magnet. The internal structure of the system is illustrated in FIG. 6B. As described above, a pinch-solenoid valve and a system power supply connected to the valve are included inside the case in which the upper module and the lower module are formed, and a tubing pump capable of controlling the transfer and fluid of the sample is provided. Cool the heat generated when driving. The upper module and the lower module are shown in more detail in FIG. 6c.

The upper surface of the driven system is shown in FIG. 7A. As shown in FIG. 7A, the sample reservoir is positioned on the left side of the system so that the syringe containing the sample can be mounted, and the pinch-solenoid valve can control the flow of fluid in the channel. It was. The tubing pump was connected to a fluid transfer air injection tube to transfer a fluid sample to the microfluidic chip or to automatically transport a buffer solution. Also, the right side of the system is shown in FIG. 7B, and a schematic diagram of the manufactured system is shown in FIG. 8.

Example 3. Sample Preparation

The microfluidic chip manufactured in Example 1 was fixed to the system of Example 2 so that the permanent magnet was located at the bottom of the chamber part. The microfluidic chip can be moved by one-axis control in the z-section direction. The inlet of the chip is connected to the tubing and all samples are controlled by the tubing pump, pinch-solenoid valve and film valve. The heater located at the top of the chamber was operated to maintain the temperature of 65 ℃ necessary for nucleic acid purification.The sample storage cartridge, which consists of the system housing part and the removable part, was equipped with a pathogen-immune nanoparticle mixed sample, lysis-binding. Buffer, washing buffer, elution buffer was filled.

Sample pretreatment is shown in FIG. 9. Pathogen concentration, washing to remove impurities, nucleic acid purification and extraction process. First, immunomagnetic nanoparticles having an antibody bound to the surface of magnetic nanoparticles are synthesized and mixed with a sample containing a pathogen. Pathogens bind to antibodies on the surface of magnetic nanoparticles. In this example, the Escherichia coli strain O157: H7 was used.

After the magnetic silica beads that can be combined with the nucleic acid in the chamber was filled, the pathogen concentration process was performed. The sample containing the pathogen-immunomagnetic nanoparticles was injected at a flow rate of 2 mL / min using a tubing pump from the sample storage cartridge, and the concentration process was performed by the magnetic field of the permanent magnet. Finally, pathogen-immunogenic nanoparticles were present in 20 μL sample.

Next, 50 μL of lysis-binding buffer was injected into the chamber and blocked with a film valve, and nucleic acid extraction from the pathogen was smoothly performed using a vibration motor for 5 minutes. The nucleic acid was extracted and bound to the magnetic silica particles filled in the chamber due to the cation bridge of the lysis-binding buffer. After washing all the lysis-binding buffer in the chamber, 40 μL of RNase free water (elution buffer) was removed. Injected.

Thereafter, the chamber was heated at 65 ° C. for 10 minutes with a heater, and the nucleic acid was separated from the magnetic silica particles. Then nucleic acid samples of concentrated pathogens were obtained in 40 μL.

With respect to the obtained sample, gel electrophoresis was carried out using a raw material before concentration of E. coli O157: H7 strain, a sample obtained from a microfluidic chip after concentration of pathogens and nucleic acid purification (concentration-nucleic acid purification liquid) through nucleic acid amplification technique (PCR). The comparison is shown in Figure 10 (M: DNA 100 bp marker, 10 ³ -1: 10 ³ -1 E. coli O157: H7 sample concentration of CFU / mL).

As a result, as can be seen in Figure 10, using a nucleic acid amplification technology can be confirmed that the detection after on-chip bacterial enrichment and nucleic acid purification at a concentration of 10 ² -1 CFU / mL that can not be detected.

In addition, the results obtained by confirming the effect of enrichment and purification of the raw material before the concentration of the E. coli O157: H7 strain and the sample obtained from the pathogen concentration and nucleic acid purification system (concentration-refining solution) using quantitative nucleic acid amplification technology (quantitative PCR). Shown in As can be seen in Figure 11, higher fluorescence intensity was observed in the sample subjected to the concentration-purification process, it can be seen that the concentration of the pathogen was efficiently achieved through the concentration-purification process.

In addition, as a result of analyzing the nucleic acid amplification starting point (Cq value) of the raw material and the sample subjected to the concentration-purification, as shown in Table 1 below, it shows a significantly lower level of Cq value as compared to the raw material. It was confirmed that the purification method showed an excellent nucleic acid purification effect.

Concentration (CFU / mL) Raw Material (Cq) Concentrated-Refined Liquid (Cq) 10 ³ 30.82 24.02 10 ² 35.22 27.74 10 42.53 28.97 One - 30.70

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (9)

A pretreatment system for concentrating and purifying samples,
The pretreatment system comprises a sample storage cartridge; A pinch-solenoid valve system in communication with said sample storage cartridge; A lower module connected to the valve system; An upper module positioned above the lower module; A microfluidic chip mounted on the lower module; And a tubing interlocking pump connected to the microfluidic chip,
The sample storage cartridge includes a plurality of cartridges for storing a sample and a buffer,
The pretreatment system is characterized in that the concentration and purification of the sample is automated, the pretreatment system for the concentration and purification of the sample.
The method of claim 1,
The sample is a pathogen; And magnetic nanoparticles having an antibody bound to the surface thereof, the pretreatment system for concentrating and purifying a sample.
The method of claim 1,
The sample storage cartridge is characterized in that for storing the sample, lysis buffer, elution buffer and wash buffer, the pretreatment system for the concentration and purification of the sample.
The method of claim 1,
The lower module is characterized in that the permanent magnet and the electric motor is provided, the pretreatment system for the concentration and purification of the sample.
The method of claim 1,
The upper module is provided with a film valve actuator for operating the film valve of the heater and the microfluidic chip, the pretreatment system for the concentration and purification of the sample.
The method of claim 1,
The microfluidic chip is a substrate; A sample injection hole formed on the substrate; A channel forming a movement path of the sample; A purification chamber connected to the channel; An impurity removal outlet through which impurities removed from the purification chamber are discharged; An air inlet through which air is injected into the channel; And a purified nucleic acid outlet through which the purified sample exits the purification chamber.
The method of claim 6,
The sock end of the purification chamber is characterized in that the film valve is provided, the pretreatment system for the concentration and purification of the sample.
The method of claim 6,
A permanent magnet is provided at the bottom of the purification chamber, the pretreatment system for the concentration and purification of the sample.
The method of claim 6,
The microfluidic chip further comprises a vibration motor, the pretreatment system for the concentration and purification of the sample.

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