KR20230116635A - Microfluidic device-based apparatus and method for pretreating biofluidic sample - Google Patents

Abstract

The present invention is an apparatus for preprocessing a biofluid sample, including a sample inlet, a medium inlet, a central fluid channel, a removal outlet, and a sample collection unit, wherein a first connection channel is formed between the inlet and outlet and the central fluid channel, wherein the Provided is a biofluid sample preprocessing device and a biofluid sample preprocessing method using the same, characterized in that a second connection channel is formed between the central fluid channel and the sample collection unit, and a one-way valve is provided in the first connection channel. . The device includes a one-way valve, and is characterized in that a large amount of sample can be pretreated without pulsation that may affect fluid flow.

Description

Microfluidic device-based apparatus and method for pretreating biofluidic sample

The present invention relates to a microfluidic device-based biofluid sample preprocessing device and method capable of processing a large amount of samples.

In addition to normal cells, various biological fluid samples such as blood, urine, cerebrospinal fluid, and sputum contain disease-related cells such as blood cancer cells, leukocytes, fungi, bacteria, and viruses. Sample preprocessing techniques for isolating, concentrating, or washing these disease-related cells play an important role in the field of in vitro diagnostics by enabling high sensitivity and high accuracy detection.

Centrifugation has been mainly used for pretreatment of large-volume biological samples. This can cause physical damage to cells due to the effect of centrifugal force, and there are limitations such as the possibility of loss of target cells due to the need for pipetting before and after using the centrifuge.

Various microfluidic device technologies have been developed to overcome the limitations of the existing centrifugal separation methods. Methods for transporting fluid within microfluidic devices are largely divided into passive fluid transport methods and active fluid transport methods, depending on whether external force is used. can

The passive fluid transport method does not use an external force, and there are methods using surface tension, pressure drive, osmotic pressure, capillary force, and hydrostatic pressure, and the active fluid transport method uses an external force. This is a method of injecting and transporting fluid by generating pressure in a microfluidic channel.

As an active fluid transfer method, a method using a syringe pump, a piezoelectric pump, or a pneumatic pump, or a method using a peristaltic pump in particular is used. The peristaltic pump has the advantage of being suitable for handling large-volume samples by continuously rotating, but pulsation inevitably occurs in the flow generated due to the characteristics of the operating mechanism in which the roller rotates and pushes the fluid in the tubing by pressing the tubing. There is a problem that greatly affects the flow phenomenon in the microfluidic device.

Therefore, in order to minimize the pulsation caused by the peristaltic pump, a one-way valve such as a check valve for preventing backflow was required, but the check valve has moving parts, so there is a possibility of failure and separate management is required.

Korean Patent Publication No. 2014-0092496

For this reason, the present invention is to provide a microfluidic device-based biofluid sample preprocessing device and preprocessing method including a Tesla valve that can act as a one-way valve without additional moving parts.

However, the problem to be solved by the present invention is not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

The present invention is a preprocessing device for a biological fluid sample,

a sample inlet, a medium inlet, a central fluid channel, a purge outlet and a sample collection;

A first connection channel is formed between the inlet and outlet and the fluid channel,

A second connection channel is formed between the central fluid channel and the sample collection unit;

Provided is a biofluid sample preprocessing device, characterized in that a one-way valve is provided in the first connection channel.

In one embodiment of the present invention, the one-way valve is characterized in that the Tesla valve.

In another embodiment of the present invention, the pretreatment device,

The sample injected into the sample inlet forms a core flow in the direction of the removal outlet,

The medium injected into the medium inlet may form a side flow toward the sample collection outlet.

In another embodiment of the present invention, the one-way valve,

It is characterized in that the fluid can flow in one direction from the sample inlet and the medium inlet to the central fluid channel through the first connection channel.

In another embodiment of the present invention, the one-way valve,

It is characterized in that it can flow in one direction from the central fluid channel to the removal outlet through the first connection channel.

In another embodiment of the present invention, the sample is characterized in that it includes an analyte and a viscoelastic fluid.

In another embodiment of the present invention, the medium is characterized in that a cell culture medium or a buffer solution.

In addition, the present invention includes the steps of injecting a sample into the sample inlet and injecting the medium into the medium inlet (S1);

moving the sample and medium through a first connection channel equipped with a one-way valve and passing through a central fluid channel (S2); and

A biofluid sample pretreatment method using the device according to the present invention is provided, including the step (S3) of collecting the pretreated sample from the sample collection outlet.

The present invention includes a Tesla valve, which is a one-way valve that can be operated using only the structure of a microfluidic channel, in the device, thereby minimizing pulsation caused by the operation of a moving part such as a peristaltic pump. A large-capacity biofluid sample can be efficiently pretreated without affecting the flow phenomenon in the device.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention described in the claims.

1 is a diagram showing a biofluid sample preprocessing device according to an exemplary embodiment of the present invention.
2 is a diagram illustrating a one-way valve according to an exemplary embodiment of the present invention.
3 is a diagram illustrating a separation and cleaning technique in a fluid channel according to an exemplary embodiment of the present invention.
Figure 4 is a diagram showing the results of comparing the pulsation of a device including a one-way valve and a device without a one-way valve of the present invention.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, since various changes can be made to the embodiments, the scope of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents or substitutes to the embodiments are included within the scope of rights.

Terms used in the examples are used only for descriptive purposes and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't

In addition, in the description with reference to the accompanying drawings, the same reference numerals are given to the same components regardless of reference numerals, and overlapping descriptions thereof will be omitted. In describing the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description will be omitted.

Throughout the specification, when a member is said to be located “on” another member, this includes not only a case where a member is in contact with another member, but also a case where another member exists between the two members.

Throughout the specification, when a certain component is said to "include", it means that it may further include other components rather than excluding other components.

The sample described herein refers to a biological sample obtained from an animal, preferably a mammal, and most preferably a human, and if a microfluidic device can be applied in various fields such as chemistry, biotechnology, and medicine, can be selected without limitation. For example, it may be cell fluid, blood, virus, bacteria, cells, low-concentration disease cells, etc., and the analyte included in the sample may have a nano-size and/or a micro-size.

As used herein, 'pretreatment' refers to a process of processing a sample so that it can be easily used for diagnosis, prognosis, or other various biological analyses, and is separated to include only the analyte, washed, or It may refer to each process of enrichment or a process performed by including two or more of the above actions. For example, it may refer to a process of isolating and concentrating only substances that can be used for diagnosis of diseases, that is, cancer cells, leukocytes, fungi, bacteria, and viruses, from a biological sample including normal cells obtained from an individual.

Hereinafter, as a preprocessing device for a biofluid sample of the present invention,

a sample inlet, a medium inlet, a central fluid channel, a purge outlet and a sample collection;

A first connection channel is formed between the inlet and outlet and the central fluid channel,

A second connection channel is formed between the central fluid channel and the sample collection unit;

A biofluid sample preprocessing device, characterized in that a one-way valve is provided in the first connection channel, will be described in detail with reference to embodiments and drawings. However, the present invention is not limited to these examples and drawings.

As shown in FIG. 1, the present invention is a preprocessing device 100 for a biofluid sample,

It includes a sample inlet 110, a medium inlet 120, a fluid channel 130, a removal outlet 140 and a sample collection unit 150,

A first connection channel 161 is formed between the inlet and outlet and the fluid channel,

A second connection channel 162 is formed between the central fluid channel and the sample collection unit,

It corresponds to the biofluid sample preprocessing device 100, characterized in that the one-way valve 170 is provided in the first connection channel.

In addition, the present invention includes the steps of injecting a sample into the sample inlet and injecting the medium into the medium inlet (S1);

moving the sample and medium through a first connection channel equipped with a one-way valve and passing through a central fluid channel (S2); and

This corresponds to a biofluid sample pretreatment method using the device according to the present invention, including collecting the preprocessed sample from the sample collection unit (S3).

In the method, at the same time as step S3, residues and interfering substances unnecessary for analysis may be separated from the removal outlet and then removed.

In the device of the present invention, the sample inlet, the medium inlet, the central fluid channel, the first connection channel, the second connection channel, the removal outlet, and the sample collection unit may be formed on a plate-shaped substrate. The sample inlet, the medium inlet, the fluid channel, the removal outlet, and the sample collection unit are all organically connected by a first connection channel or a second connection channel, and are microfluidic devices.

The substrate is polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), cyclic olefin copolymer (COC), polyacrylates, polycarbonates , polymer materials such as polycyclic olefins, polyimides, and polyurethanes may optionally be used.

More specifically, in the device, the sample inlet 110 is an inlet into which a sample requiring pretreatment is introduced, and the medium inlet 120 is an inlet into which a medium to be used for pretreatment is introduced. As shown in FIG. 1, the medium inlet is branched at the inlet and formed around the sample inlet, and the branch is connected again in a passage connected from the sample inlet, through which the fluid channel 130 is formed at the first branch point 181. connected with The sample and medium passing through the fluid channel 130 diverge at the second branch point 182 at the end of the fluid channel 130, and the fluid passing through the first connection channel goes to the removal outlet 140, and the second connection channel The fluid passing through may be collected through the sample collection unit 150 .

According to one embodiment of the present invention, as shown in FIG. 2, a plurality of one-way valves 160-1 and 160-2 are provided in the first connection channel between the inlet and outlet and the fluid channel. The one-way valve is a Tesla valve, and the Tesla valve 160-1 allows flow in one direction from the sample inlet and the medium inlet to the central fluid channel through the first connection channel, and the Tesla valve 160-2 ) is allowed to flow in one direction from the central fluid channel to the removal outlet through the first connection channel.

As shown in FIG. 2, the Tesla valve operates as a check valve and has a structure in which loop structures are connected. The loop structure has a rounded end (a) and a sharp end (b), wherein (a) is located in the inlet direction (upstream of the sample) and (b) is disposed in the outlet direction (downstream of the sample) . The Tesla valve allows the sample and the medium to flow in one direction from the upstream, which is an inlet, to the downstream, which is an outlet, and can be moved through the path shown in FIG. 2(c).

The pulsation damping rate of the flow varies according to the number of loop structures included in the Tesla valve or according to the shape and size of the channels of the loop structure. Therefore, the optimal number of loop structures required for device operation can be obtained by evaluating the pulsation damping rate per unit number or per unit area. For example, in the case of a straight microfluidic channel without a Tesla valve loop structure and a microfluidic channel designed with one Tesla valve loop structure, assuming that the degree of pulsation of the channel without a Tesla valve is 100, the Tesla valve loop structure When designing one, it was confirmed that about 10% of the pulsation was attenuated. In this case, assuming that the attenuation rate according to the number of Tesla valve loop structures is directly proportional, having at least 10 loop structures will eliminate pulsation in the microfluidic channel.

According to one embodiment of the present invention, in the pretreatment device, the sample injected into the sample inlet forms a core flow in the direction of the removal outlet, and the medium injected into the medium inlet forms a side flow toward the sample collection unit. to be The sample includes an analyte and a viscoelastic fluid, and the medium includes a cell culture medium or a buffer solution.

The analyte is a disease-related cell, and the disease may be cancer. The cell type includes, for example, male hormone-dependent prostate cancer cell line LNCaP cells, hormone-independent prostate cancer cell lines PC-3 and DU-145 cells, breast cancer cell line MCF-7 cells, lung cancer cell line A549 cells, NCI- H292 cells, K-562 cells, a chronic myelogenous leukemia cell line, HeLa cells, a cervical cancer cell line, HepG2 cells, a liver cancer cell line, or P388 cells, a lymphatic cancer cell line. However, the types listed above are examples, and are not limited thereto as long as they are substances that can be used for diagnosis of diseases.

More specifically, as shown in FIG. 3, the device of the present invention isolates disease-related cells, which are analyte substances, using a viscoelastic fluid, exchanges the medium (medium) with another fluid, and then collects the cells after passing through the device to wash them. can be used to increase the analytical sensitivity.

The viscoelastic fluid may have a viscosity of 1 to 20 cP (centi-poise), and as a polymer material, polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), polyacrylamide ( It may include one or more selected from the group consisting of polyacrylamide (PAA), deoxyribonucleic acid (DNA), xanthan gum, and hyaluronic acid (HA).

The medium may include a buffer solution or culture medium. The buffer may include phosphate buffered saline (PBS), Tris-buffered saline (TBS), HEPES-buffered saline (HBS), and the like, and the culture medium may include MEM, DMEM, RPMI 1640, or IMDM. can

In the device of the present invention, a sample containing an analyte requiring pretreatment such as separation and washing suspended in a viscoelastic fluid harmless to the human body is injected into the sample inlet (core-flow) in the center of the microfluidic chip, and a pure buffer or cell culture medium is injected. can flow toward the channel wall and form a co-flow according to the characteristics of laminar flow by injecting it into a medium inlet that can form a side flow.

Thereafter, by using the flow characteristics of the viscoelastic fluid, the target cells suspended in the biofluid sample may be laterally moved toward the wall of the channel to be separated and washed.

That is, the present invention applies the micro-Tesla structure to the microchip design in order to eliminate pulsation in the fluid channel, the Tesla valve can enable stable flow control, and the sample inlet and the medium inlet have a tubing pump Since the micro-Tesla structure moves the sample in one direction without an external device, the influence of external pulsation can be minimized.

More specifically, in the fluid channel, cell separation and washing are performed based on coaxial flow using a viscoelastic fluid. Due to the medium injected through the medium inlet forming the side flow on the channel wall, the mixture containing normal and abnormal cells suspended in the viscoelastic fluid concentrates and flows in the center of the fluid channel, and at this time, the cells in the flow flow in the fluid. It is simultaneously affected by the hydrodynamic force, inertial force and elastic force acting by the pressure gradient, and as a result, the disease-related cells, which are the objects of separation and washing, are moved to other fluids on the channel wall side and exchanged with the medium to the side outlet. collection is possible. Other residues and interfering substances unnecessary for analysis can still flow to the center of the channel and be removed toward the removal outlet located in the middle.

[Example]

Example 1. Large-capacity sample pretreatment method

Pretreatment of blood samples was performed using the apparatus shown in FIG. 1 . After connecting the tubing connected to the peristaltic pump to the sample inlet and the medium inlet of the device, leukocytes and disease-related cells suspended in a 1000 ppm polyethylene oxide (PEO) solution, a viscoelastic fluid, were placed in the sample inlet, prostate cancer cells, and DU. A sample in which -145 cells were suspended was injected, and phosphate buffered saline (PBS) was injected into the medium inlet. At this time, the flow rate of the sample and medium, respectively, was injected through a tubing pump at 10 μl/min and 100 μl/min. The Tesla valve designed and manufactured in the first connection channel connected to the inlet side not only prevents backflow that can occur due to peristaltic tubing pumps, but also minimizes pulsation, which is suitable for cell separation and washing based on the flow characteristics of viscoelastic fluids. It was advantageous.

After injection, each cell could be separated and washed based on the coaxial flow-based flow characteristics of the viscoelastic fluid, as shown in FIG. 3 . Specifically, relatively large DU-145 cells (12-20 μm) are pushed toward the medium (PBS) by the force acting laterally in the viscoelastic fluid, and relatively small white blood cells (9-14 μm) remains in the viscoelastic fluid and flows into the outlet connection channel. Through this, after the injection was completed, the pre-treated sample was collected in the sample collection unit, and as a result of checking the pre-treated sample, most of the white blood cells that hindered the analysis were removed through the removal outlet, which is the central exit, and the cells to be analyzed, prostate cancer cells DU It was confirmed that the -145 cells were separated and washed through the sample collection part, which is a side exit. In order to confirm cell separation more clearly, it can be confirmed by fluorescence staining through a biomarker, and leukocytes were stained with CD 45 and DU-145 with EpCAM.

Example 2. Pulsation evaluation of a biofluid sample pretreatment device

In this embodiment, it was confirmed how the effect of pulsation generated by the tubing pump is minimized in the fluid channel including the Tesla valve as in the present invention to ensure flow stability.

The flow rate was evaluated according to the presence or absence of the micro-Tesla structure in the fluid channel, and is shown in FIG. 4 . A tubing pump was used to control the flow rate, and the flow coming out of the outlet of the microchannel was measured using a flow sensor.

As can be seen in FIG. 4, when checked in the two flow rate ranges, the pulsation of the flow rate is lowered when the micro-Tesla structure is present (w / Tesla) compared to the case without the micro-Tesla structure (w / o Tesla). Confirmed.

As described above, although the embodiments have been described with limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, the described techniques may be performed in an order different from the method described, and/or components of the described system, structure, device, circuit, etc. may be combined or combined in a different form than the method described, or other components may be used. Or even if it is replaced or substituted by equivalents, appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents of the claims are within the scope of the following claims.

100: biofluid sample pre-processing device
110: sample inlet 120: medium inlet
130: fluid channel 140: removal outlet
150: sample collection unit 161: first connection channel
162: second connection channel 170-1, 170-2: one-way valve
181: first branch point 182: second branch point

Claims (8)

As a pre-processing device for a biofluid sample,
a sample inlet, a medium inlet, a central fluid channel, a purge outlet, and a sample collector;
A first connection channel is formed between the inlet and outlet and the central fluid channel,
A second connection channel is formed between the central fluid channel and the sample collection unit;
A biofluid sample preprocessing device, characterized in that a one-way valve is provided in the first connection channel.
According to claim 1,
The one-way valve is a Tesla valve, characterized in that, the biofluid sample preprocessing device.
According to claim 1,
The pre-processing device,
The sample injected into the sample inlet forms a core flow in the direction of the removal outlet,
The biofluid sample preprocessing device, characterized in that the medium injected into the medium inlet forms a side flow in the direction of the sample collection unit.
According to claim 1,
The one-way valve,
A biofluid sample preprocessing device, characterized in that it allows the flow in one direction from the sample inlet and the medium inlet to the fluid channel through the connection channel.
According to claim 1,
The one-way valve,
A biofluid sample preprocessing device, characterized in that it allows the fluid to flow in one direction from the fluid channel to the removal outlet through the connection channel.
According to claim 1,
Wherein the sample comprises an analyte and a viscoelastic fluid.
According to claim 1,
The medium is a cell culture medium or a buffer solution, characterized in that, the biofluid sample preprocessing device.
Injecting a sample into a sample inlet and injecting a medium into a medium inlet (S1);
The sample and medium are moved through a first connection channel equipped with a one-way valve and pass through a central fluid channel (S2); and
A method for pre-processing a biofluid sample using the device of claim 1, comprising the step (S3) of collecting the pre-processed sample from the sample collection unit.