KR102373309B1 - Microfluidic device and radioimmunoassay method using the same - Google Patents

Abstract

The present invention relates to a microfluidic device and a radioimmunometry method using the same, and more particularly, to a lower housing; an upper housing engageable with the lower housing; and a fluid channel disposed between the lower housing and the upper housing, wherein the fluid channel includes a liquid inlet connected to one end of the fluid channel, a gas inlet communicating with the fluid channel for gas input, and a patterned fluid channel To a microfluidic device comprising a sensing unit comprising: and a fluid outlet connected to the other end of a fluid channel;

Description

Microfluidic device and radioimmunoassay method using the same

The present invention relates to a microfluidic device and a method for measuring radioimmunity using the same, and more particularly, to a microfluidic device with improved reaction efficiency and particularly suitable for radioimmunometry, and a method for measuring radioimmunity using the same.

As a means of primarily acquiring a large amount of biological information, biological detection technology is very important in industrial applications such as disease prevention and diagnosis. In order to proceed with systematic biological research, a tool that can simultaneously compare and analyze large amounts of samples within a short time is needed. Recently, the need to develop a microscopic bioanalysis system in the bio industry has great significance in genome and proteomic research and drug development.

Immunodiagnosis is an in vitro diagnostic method that qualitatively or quantitatively measures disease factors using biomarkers present in blood or urine, and an enzyme-linked immunosorbent assay (ELISA) using enzymes as a marker is representative. On the other hand, radioimmunoassay (RIA) is an immunodiagnostic method using radioactive isotopes as markers and has been used to diagnose diseases by quantifying the concentrations of trace amounts of hormones, physiologically active substances, and drugs.

For example, Korean Patent Application Laid-Open No. KR 10-2004-0044839A discloses a radioimmunoassay method for measuring the concentration of macrophage migration inhibitory factors. It is used in the diagnosis of many diseases, such as cancer. However, there are restrictions on use due to the nature of using radioactive materials, and in particular, a lot of radioactive waste is generated during measurement procedures such as sample preparation and cleaning, and radiation exposure of workers performing measurements may occur.

As a method for performing the microbiological detection method, there is a method of using a microdevice in which an antibody is fixed inside a microfluidic channel. At this time, a method of immobilizing an antibody on the surface of the channel by coating, etc. The apparatus is a miniature device for handling trace amounts of fluids manufactured using microfabrication technology for this purpose. It can be designed in various ways, can handle a small amount of fluid, and has a large ratio of surface area to volume, so it is widely applied in biological diagnosis.

Therefore, it reduces the time required for measurement, increases analysis efficiency and reduces costs, generates less waste, has a fast diagnosis speed, reduces radiation exposure of workers when performing radioimmunometry, and can be directly applied to conventional gamma measuring instruments. If a microfluidic device and a radioimmunoassay method using the same are provided, it is expected to be widely used in related fields.

One aspect of the present invention is to provide a microfluidic device that generates less waste, has a fast diagnosis rate, and has excellent sensitivity.

Another aspect of the present invention is to provide a method for measuring radioimmunity that can be directly applied to a conventional gamma meter while having a fast diagnosis speed and a small amount of radiation exposure of workers when performing radioimmunometry.

Accordingly, according to one aspect of the present invention, it includes a body having a fluid channel formed therein, wherein the fluid channel includes a liquid inlet connected to one end of the fluid channel, a gas inlet communicating with the fluid channel for gas input, and a patterned fluid channel A microfluidic device is provided, comprising a sensing unit comprising: and a fluid outlet connected to the other end of the fluid channel.

According to another aspect of the present invention, the method comprising: introducing a liquid sample including a radioisotope-labeled analyte to the liquid inlet of the microfluidic device; And, there is provided a radioimmunoassay method comprising the step of introducing a gas into the gas inlet.

According to the present invention, the time required for measurement is reduced, the analysis efficiency is increased, the cost is reduced, the generation of waste is small, the diagnosis speed is fast, the radiation exposure of the operator is small when performing radioimmunometry, and it is directly applied to the conventional gamma meter Provided are a microfluidic device capable of doing this and a radioimmunometry method using the same.

1 schematically shows an exemplary microfluidic device of the present invention.
2 is a result of measuring radioactivity after radioimmunometry using a microfluidic device in Examples 1 to 4;
3 is a result of evaluating the presence or absence of contamination of the test tube by the microfluidic device to which the isotope-labeled antigen is attached according to Experimental Example 2.
4 is a photograph taken using an optical microscope (ECLIPSE Ts2, Nikon) of the liquid and gas section in the fluid channel after the liquid sample and gas input are completed in Example 5. The ratio of the liquid and gas section in the fluid channel is about It can be seen that 1:1 was formed.
5 is a result showing the degree of attachment of the isotope-labeled protein according to the presence or absence of gas injection according to Experimental Example 3.
6 is a photograph of a microfluidic device having an exemplary stacked structure of the present invention.
7 is a result showing the degree of amplification of a sensing signal according to the number of layers of microfluidic channels according to Experimental Example 4.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiment of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

According to the present invention, the amount of sample required for diagnosis can be reduced, the sensitivity is excellent, the radiation exposure of workers when performing radioimmunometry is small, and a microfluidic device that can be directly applied to a conventional gamma meter and radioimmunity using the same A measurement method is provided.

More specifically, the microfluidic device of the present invention includes a body in which a fluid channel is formed, wherein the fluid channel includes a liquid inlet connected to one end of the fluid channel, a gas inlet communicating with the fluid channel for gas input, and a patterned body. It will include a sensing unit made of a fluid channel and a fluid outlet connected to the other end of the fluid channel.

In the present invention, it is understood that the 'microfluidic device' can be used interchangeably with the 'microfluidic device'. It refers to an inducible device.

The present invention relates to a plastic (polymer)-based microfluidic device, and the material of the body that can be used in the present invention is polydimethylsiloxane (PDMS), polycarbonate (PC), polystyrene (PS), polypropylene (PP) ) and the like, or by using polymethyl methacrylate (PMMA), cyclic olefin copolymer (COC), etc. may be produced for one-time use, but is not particularly limited thereto.

In the present invention, the body in which the fluid channel is formed may be formed of, for example, a lower housing, an upper housing engageable with the lower housing, and a fluid channel disposed between the lower housing and the upper housing, but is not limited thereto, It may also be manufactured by a 3D printing technique or the like. As such, the manufacturing process of the microfluidic device of the present invention is not particularly limited, and when the injection molding method is used, a polymer is generally injected into the mold at high temperature using a mold and/or a mold, cooled, and then separated from the mold. method is being used. At this time, the reverse image of the mold is formed on the plastic surface, and the device and the lower plate derived therefrom are combined and sealed to manufacture a product.

When the microfluidic device is manufactured, the bonding method of each housing or the bonding of each layer constituting the body is not particularly limited when the body is formed as a laminate. For example, thermal bonding, plasma bonding, ultrasonic bonding, optical method, etc. can be used. there is.

In the present invention, "sample" or "sample" is not particularly limited as long as it can contain a sample to be detected, that is, an analyte. Illustratively, the sample may be a biological sample, eg, a biological fluid or biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid, and the like. Biological tissue is an agglomeration of cells, and is a collection of intracellular substances and specific types of substances that generally form one of the structural substances of human, animal, plant, bacterial, fungal or viral structures, including connective tissue, epithelial tissue, muscle tissue, and nerve. This may be an organization or the like. Examples of biological tissue may also include organs, tumors, lymph nodes, arteries, and individual cells.

Meanwhile, the "analyte", which is an analyte, may be understood to mean a molecule or other substance in a sample to be detected. For example, the sample may include antigenic substances, ligands (mono- or polyepitopes), haptens, antibodies, and combinations thereof. Specifically, the sample may be, for example, a toxin, an organic compound, a protein, a peptide, a microorganism, an amino acid, a nucleic acid, a hormone, a steroid, a vitamin, a drug, a drug intermediate or by-product, a bacterium, a viral particle, a yeast, a fungus, a protozoan and the above. It may include, but is not limited to, metabolites of substances or antibodies thereto. However, in general, it may be an antigen or an antibody. In this regard, exemplary subjects include ferritin; creatinine kinase MB (CK-MB); digoxin; phenytoin; phenobarbitol; carbamazepine; vancomycin; gentamicin; theophylline; valproic acid; quinidine; luteinizing hormone (LH); follicle stimulating hormone (FSH); estradiol, progesterone; C-reactive protein (CRP); lipocalin; IgE antibody; cytokines; vitamin B2 micro-globulin; glycated hemoglobin (Gly.Hb); cortisol; digitoxin; N-acetylprocainamide (NAPA); procainamide; antibodies to rubella, such as rubella-IgG and rubella IgM, for example antibodies to toxoplasmosis, such as tox (toxo-IgG) and toxoplasmosis IgM; testosterone; salicylate; acetaminophen; hepatitis B virus surface antigen (HBsAg); antibodies to hepatitis B core antigens such as anti-hepatitis B core antigens IgG and IgM (anti-HBC); human immunodeficiency virus 1 and 2 (HIV 1 and 2); human T-cell leukemia virus 1 and 2 (HTLV); hepatitis B e antigen (HBeAg); antibodies to hepatitis B e antigen (anti-HBe); influenza virus; thyroid stimulating hormone (TSH); thyroxine (T4); total triiodothyronine (total T3); free triiodothyronine (free T3); Carcinogenous Embryonic Antigen (CEA); lipoproteins, cholesterol, and triglycerides; and alpha fetoprotein (AFP). Drugs of misuse and control substances, specifically amphetamines; methamphetamine; barbiturates such as amobarbital, secobarbital, pentobarbital, phenobarbital and barbital; benzodiazepines such as Librium and Valium; cannabinoids such as Hashish and Marijuana; cocaine; fentanyl; LSD; metaqualone; opiates such as heroin, morphine, codeine, hydromorphone, hydrocodone, methadone, oxycodone, oxymorphone and opium; phencyclidine; and propoxyhen, but is not limited thereto.

In the present invention, as a labeling substance, metal colloids such as colloidal gold, colloids of nonmetals such as selenium colloid, colored resin particles, dye colloids, insoluble particulate substances such as colored liposomes, alkaline phosphatase, and enzymes that promote a color reaction such as peroxidase , a fluorescent dye, a radioisotope, etc. can be exemplified, and a radioisotope is preferably used. The radioisotope may include, for example, ¹⁴ C, ³ H, ¹²⁵ I, ³³ P, and ³⁵ S, but is not limited thereto.

Meanwhile, in the present invention, the fluid channel is connected to a liquid inlet connected to one end of the fluid channel, a gas inlet communicated with the fluid channel for gas input, a sensing unit comprising a patterned fluid channel, and the other end of the fluid channel. It will include a fluid outlet.

A sample including a sample is put into the liquid inlet, the gas inlet communicates with a fluid channel for gas input, such as air, and the sensing unit consists of a patterned fluid channel, and the inputted fluid is discharged through the fluid outlet .

At this time, the liquid inlet, the sensing unit, and the fluid outlet are sequentially arranged along the fluid channel, and the gas inlet is in communication with a gas input line joined to the liquid input line, which is a fluid channel between the liquid inlet and the sensing unit. Alternatively, the liquid inlet, the gas inlet, the sensing unit, and the fluid outlet may be sequentially disposed along the fluid channel.

In the microfluidic device of the present invention, the 'liquid input line (liquid flow pipe)' specifically refers to a fluid channel between the liquid inlet and the sensing unit, and the 'gas input line (gas flow pipe)' is the gas inlet and the liquid input line. It specifically refers to a fluid channel between the two, and more specifically, the gas input line refers to a line through which gas is input from the gas inlet to the junction of the liquid input line.

The step of introducing the gas through the gas inlet may be performed simultaneously with or alternately with the step of introducing the liquid sample into the liquid inlet, and the liquid sample generates turbulence in the fluid channel of the sensing unit by the introduction of the gas. It is possible to increase the amount of antigen and antibody adhered to the channel surface by the stirring effect derived from the formation. Fluid flow inside the microfluidic channel is generally formed as a laminar flow, and thus antigens and antibodies in the liquid sample that can adhere to the channel are limited to those present on the surface. However, the input of the gas provided in the present invention forms turbulence in the channel due to the difference in viscosity, which is a unique characteristic of the fluid, and through this, the antigen and antibody present in the liquid sample can be uniformly attached to the surface of the channel. do.

In order to maintain a constant linear velocity in the microtubule, it is preferable that the width of the microtubule is constant, and in particular, it is preferable that the width of the part from the intersection of the liquid flow pipe and the gas flow pipe to the outlet of the microtubule be uniform. For example, the microtubule may be a tube having a width of 50 to 200 μm, and the shape of the cross-section is not limited, but may be, for example, a circle or a square. Meanwhile, the width of the liquid flow tube and the gas flow tube among the microtubules may be the same or different, for example, the widths of the liquid flow tube and the gas flow tube may be 100 and 50 μm, respectively. At this time, the liquid sample is, for example, input at a flow rate of 1 to 100 μL/min into the liquid inlet, and the gas is preferably introduced at a flow rate of 1 to 200 μL/min into the gas inlet, as a result, for example, The ratio of the length of the gas section to the liquid section in the microtubule is 1:0.25 to 1.5.

When the ratio of the length of the gas section to the liquid section is less than 1:1.5, the fluid flow in the channel exhibits a laminar flow, so there is a problem that the stirring effect does not appear, and when it exceeds 1:0.25, the excessive rotation of the fluid (over -twirling), there is a problem that the protein present in the liquid sample does not adhere to the surface of the microtubule.

After the input of the liquid sample and the gas is completed, the ratio of the length of the liquid and gas section in the sensing unit is preferably 1:0.25 to 1.5, for example, 1:0.3 to 1.25, preferably about 1:1 .

On the other hand, when the length of the liquid section is excessively long compared to the width of the fluid channel, it is difficult to have a stirring effect through gas input. Therefore, it is preferable that the ratio of the width of the fluid channel to the length of the liquid section is 1:1 to 1:7.

The inside of the microfluidic channel is an amine group, a carboxyl group, a thiol group ( Various cross-link groups can be covalently bonded to each of these functional moieties by using thiol) or aldehyde groups, hydroxyl groups, and the like. For example, as a material capable of binding to an amine group, aldehyde, acyl azide, carbonate, carbodiimide, isothiocynate, NHS ester , cyanogen bromide, sulfonyl chloride, tosyl chloride, and the like. Binding of biomaterials can be induced by using the above-described compounds. Preferably, the inside of the microfluidic channel is surface-treated with at least one residue of an amine group, a carboxyl group, a thiol group, an aldehyde group, and a hydroxyl group, and then the antibody is it will be attached

However, depending on the degree of hydrophilicity of the plastic material forming the microfluid transfer space, hydrophobic materials, for example, hydrophobic polytetrafluoroethylene (PTFE)-based polymer, parylene-based polymer, fluorine (F) ) functional group, a compound having a chlorine functional group (Cl), etc., a monomer, a dimer, a polymer, or nanoparticles can be used alone or in combination to control the flow characteristics of the microfluid.

For example, a microfluidic device made of a PDMS material has hydrophobicity and requires hydrophilic modification to prevent non-specific binding of biomaterials. In addition, it is necessary to introduce a functional group that induces binding to a biological material for the attachment of the antibody to the surface of the microtubule. As an example, hydrophilic modification can be performed through O ₂ plasma treatment, and hydrophobicity can be returned over time, so it is preferable to introduce a functional group before the hydroxyl group generated by the surface treatment disappears. The hydrophilic modification may be accomplished through chemical methods such as piranha (piranha, H ₂ SO ₄ /H ₂ O ₂ ) solution treatment, NaOH/KOH solution treatment, and acid solution treatment. An amine group (-NH ₂ ) or an aldehyde group (-CHO) is generally used as a functional group for covalent bonding of the biomaterial to the surface of the microtubule. As an example, APTMS (3-aminopropyl) trimethoxysilane), which is an aminosiloxane series, is introduced into the hydrophilic microtubule by using a syringe pump to introduce an amine group. In addition, by flowing glutaraldehyde into the microtubule into which the amine group is introduced, the aldehyde group is introduced. The reaction time for introducing the functional group may vary depending on the concentration of the injection solution, and may be injected at a flow rate of 1 to 100 μL/min depending on the size of the microtubule. In addition, N-hydroxysuccinimide such as sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and a crosslinking agent having a maleimide functional group can be used to attach the thiol group of the antibody to the microtubule surface. can The antibody is attached to the surface of the microtubule by flowing an antibody that specifically binds to the antigen to be diagnosed in the microtubule into which the functional group is introduced.

As an example, when diagnosing T4 (Thyroxine) antigen, anti-T4, an antibody that specifically binds thereto, can be attached to the surface of a microtubule, and the antibody is added to PBS (phosphate) in consideration of the size of the microtubule. -buffered saline) can be diluted and used. The reaction time for antibody attachment may vary depending on the concentration of the injected antibody, and may be injected at a flow rate of 1 to 100 μL/min depending on the size of the microtubule. In the above process, unreacted regions where functional groups cannot be introduced and antibodies are not attached may occur. In order to prevent non-specific binding of proteins by this, blocking treatment is performed by flowing bovine serum albumin (BSA).

The antigen labeled with the isotope and the antigen present in the unknown sample are flowed to the surface-treated device to attach the antigen to the surface of the microtubule. As an example, I-125 isotopes are labeled with T4, an antigen to be diagnosed, using the Iodogen method. Depending on the characteristics of the antigen to be diagnosed, the type and specific activity of the labeled isotope may be different. The antigen labeled with the isotope and the antigen present in the unknown sample are injected into the microfluidic device using a syringe pump to react. The injection flow rate of the antigen can be injected at a flow rate of 1 to 100 μL/min depending on the size of the microtubule. In order to measure radioactivity in the next step, the unattached isotope-labeled antigen remaining in the microtubule must be removed, which is washed by injecting PBS. The washing time may vary depending on the amount of antigen to be injected, and may be injected at a flow rate of 1 to 100 μL/min depending on the size of the microtubule.

On the other hand, in the microfluidic device of the present invention, the sensing unit is preferably disposed so as not to overlap an imaginary straight line connecting the liquid inlet and the fluid outlet, and an exemplary arrangement structure is as shown in FIG. is disposed at one end of the microfluidic device, and the liquid inlet and the fluid outlet may be disposed at the other end of the microfluidic device.

The overall shape of the microfluidic device of the present invention is not particularly limited, and may be, for example, a rectangle, an oval, or other polygons when viewed from the top as shown in FIG. 1 . It is preferably rectangular or oval when viewed from the top.

The sensing unit may have microtubules arranged in various shapes. For example, as shown in FIG. 1 , the sensing unit may be spirally formed in a direction to maximize a surface area to increase a sensing signal.

The size of the microfluidic device of the present invention is not particularly limited, but for example, the width is 2 to 20 mm, preferably 5 to 9 mm, and the length is in the range of 3 to 75 mm, preferably 25 to 70 mm. , having a thickness of 50 μm to 14 mm, preferably 3 to 6 mm, but may be adjusted according to the size of a detection unit of a subsequently applied detection device.

Further, the body in which the fluid channel is formed may be in the form of a laminate in which two or more layers in which the fluid channel is formed are stacked, and such a structure is particularly preferable when radioimmunometry is performed using a radioisotope as a label. . In the case of microfluidic device-based immunodiagnosis using fluorescence or enzyme-linked immunosorbent assay (ELISA) as a detection signal, the signal that can be detected from the stacked microfluidic device is the top or bottom sheet by overlapping layers. It is limited to the signal emitted from However, the radioimmunoassay method using a radioisotope provided in the present invention can be amplified without overlapping signals through a stacked microfluidic device. The laminated sheet may have, for example, 2 to 5 layers, wherein the liquid inlet and the fluid outlet may be formed on the same layer, or the liquid inlet may be disposed on one outer layer and the fluid outlet may be disposed on the other outer layer, and the sensing unit may have two or more layers. It may be formed to overlap over. When the microfluidic device of the present invention is formed of a laminate, the thickness means the total thickness of the laminate.

The microfluidic device of the present invention may be for measurement by a gamma counter, a gamma spectrometer, a liquid scintillation counter, etc., and in particular, for example, for insertion of a well type detector. .

Further, according to the present invention, there is provided a radioimmunoassay method using the microfluidic device of the present invention, wherein the labeling material in the microfluidic device is a radioisotope.

More specifically, the radioimmunoassay method of the present invention comprises the steps of: introducing a liquid sample containing a radioisotope-labeled analyte to the liquid inlet of the microfluidic device of the present invention; and injecting gas into the gas inlet.

The step of injecting the liquid sample into the microfluidic device may be accomplished through a tube connection at the liquid inlet and driving of a syringe pump, and the fluid may be discharged through a tube connection at the fluid outlet.

As mentioned in relation to the microfluidic device, the step of introducing the liquid sample into the liquid inlet may be performed simultaneously with the step of introducing gas into the gas inlet, or may be performed alternately.

Preferably, the step of introducing the liquid sample into the liquid inlet and the step of introducing the gas into the gas inlet are performed simultaneously, for example, in the case of a rectangular tube having a width of 100 μm and a height of 33 μm, the liquid sample is transferred to the liquid inlet. is input at a flow rate of 1 to 100 μL/min, and the gas is input at a flow rate of 1 to 200 μL/min.

disposing the microfluidic device on a detector of an analysis device after the input of the liquid sample and the gas is completed; and confirming the result using the gamma counter.

According to the present invention, the adhesion reaction can be promoted by having a stirring effect in the microtubule through bubble formation by injection of gas.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples are only examples to help the understanding of the present invention, and the scope of the present invention is not limited thereto.

Example

Example One

After mixing the PDMS (polydimethylsiloxane) base (A) and the curing agent, PDMS (B) in a ratio of 10:1, air bubbles are removed through a vacuum pump. Pour the PDMS mixture onto the master pattern of the microfluidic device design (Fig. 1) designed for radioimmunoassay and harden at 60 °C for 1 hour. The mold is scooped out and drilled for liquid injection. A microfluidic device was manufactured by combining with the substrate by surface treatment using O ₂ plasma.

At this time, the width of the microfluidic device was 9 mm, the length was 40 mm, and the total thickness was 6 mm. is 100 μm, the width of the channel forming the gas input line from the gas inlet to joining the liquid input line was 50 μm, and the height of each channel was 33 μm. The total volume of the fluid channel was about 1.5 mm ³ , and the total surface area was 122.5 mm ² . The shape of the fluid channel was designed in a spiral design to minimize the radioactive sample (isotope-labeled antigen) required for diagnosis while maximizing the surface area to increase the detection signal (radioactivity).

In order to treat the surface of the microtubule inside the manufactured device with an antibody, a functional group capable of inducing binding to a biological material is preferentially introduced. First, for continuous infusion of the solution, connect the perforated device to the tube and syringe. 2% (3-aminopropyl)trimethoxysilane (APTMS) is flowed for 2 hours at a flow rate of 10 μL/min using a syringe pump to introduce amine groups on the surface of the microtubule. In order to remove the unreacted solution in the microtubule, it is washed by flowing ethanol at a flow rate of 20 μL/min for 10 minutes. For the introduction of the next functional group, the ethanol remaining in the microtubule is removed by blowing air. For the introduction of the aldehyde group, 2.5% glutaraldehyde (GA) is flowed at a flow rate of 10 μL/min for 1 hour. In order to remove the unreacted solution in the microtubule, it is washed by flowing distilled water at a flow rate of 20 μL/min for 10 minutes. For antibody attachment to the microtubule surface, blow off air to remove distilled water.

To attach an anti-T4 antibody that specifically reacts with an exemplary diagnostic target T4 (Thyroxine) antigen, 1 μg anti-T4/10 μL PBS (phosphate-buffered saline) antibody was added to a microtubule After injection, seal and refrigerate for 12 hours. To remove non-adherent antibodies in microtubules, wash with PBS at a flow rate of 20 μL/min for 10 minutes. In order to prevent non-specific binding of antigen in the unreacted area to which the antibody is not attached, blocking is performed by flowing 1% bovine serum albumin (BSA) at 10 μL/min for 1 hour. A commercially available radioimmunodiagnostic kit can be used for radioimmunometry using a microfluidic device, and unlabeled T4 antigen 2 contained in the radioimmunodiagnostic kit is included in the radioimmunodiagnostic kit to induce a competitive reaction on the surface of microtubules. After injecting 10 µL of the standard solution with a concentration of µg/dL into the microtubule, the reaction is stagnant for 5 minutes. Subsequently, 10 μL of a ¹²⁵ I tracer sample containing the T4 antigen labeled with ¹²⁵ I is injected into the microtubule, followed by a stagnant reaction for 5 minutes to induce a competition reaction. To remove the non-adherent antigen remaining in the microtubule, wash with PBS at 20 μL/min for 5 minutes. For radioactivity measurement in the next step, remove PBS remaining in the microtubule by blowing air.

Next, the microfluidic device was placed in a test tube and placed in a detector of a Gamma Counter (1470 WIZARD) to measure radioactivity.

Example 2

The reaction was carried out in the same manner as in Example 1, except that 10 µL of the standard solution having a concentration of 4 µg/dL of the isotope-labeled T4 antigen of Example 1 was injected into the microtubule and the reaction was stagnant for 5 minutes.

Example 3

The reaction was carried out in the same manner as in Example 1, except that 10 μL of the standard solution having a concentration of 8 μg/dL of the T4 antigen unlabeled in Example 1 was injected into the microtubule and the reaction was stagnant for 5 minutes.

Example 4

In Example 1, except that 10 μL of a quality control (QC (Shinjin Medics, RIAKEY T4 RIA Tube Ⅱ)) substance other than the standard solution of Example 1 was injected into the microtubule and stagnant reaction was performed for 5 minutes. The reaction was carried out in the same manner.

Example 5

To the microfluidic device introduced with a functional group to induce binding to the biomaterial of Example 1, 10 μL of an antibody (anti-PSA) labeled with an isotope ( ¹²⁵ I) was added to the microfluidic device at a flow rate of 1 μL/min. Antibody attachment was carried out by flowing it through the liquid inlet of the At this time, air was also introduced into the gas inlet at a flow rate of 35 μL/min. As a result, as shown in FIG. 4 , the ratio between the liquid and gas sections in the sensing unit was about 1:1. In order to remove the non-adherent antibody remaining in the microtubule, it is washed by flowing PBS at 20 μL/min for 5 minutes. For radioactivity measurement in the next step, remove PBS remaining in the microtubule by blowing air.

Next, the microfluidic device was placed in a test tube and placed in a detector of a gamma counter (Gamma Counter, 1470 WIZARD) to measure radioactivity.

Example 6

An antibody labeled with an isotope ( ¹²⁵ I) with respect to a microfluidic device as shown in FIG. 6, in which a fluid channel into which a functional group is introduced to induce binding to the biomaterial of Example 1 is stacked in three layers ( Anti-PSA) 10 μL was flowed into the liquid inlet of the microfluidic device, followed by a stagnant reaction for 5 minutes to proceed with antibody attachment. In order to remove the non-adherent antibody remaining in the microtubule, it is washed by flowing PBS at 20 μL/min for 5 minutes. For radioactivity measurement in the next step, remove PBS remaining in the microtubule by blowing air.

Next, the microfluidic device was placed in a test tube and placed in a detector of a gamma counter (Gamma Counter, 1470 WIZARD) to measure radioactivity.

comparative example One

The reaction was performed in the same manner as in Example 5, except that in Example 5, only 10 μL of a liquid sample containing an antibody was flowed without gas.

Example 7

The reaction was performed in the same manner as in Example 6, except that the microfluidic device in which the fluid channel of Example 6 was stacked in two layers was used.

Example 8

The reaction was performed in the same manner as in Example 6, except that the single-layer microfluidic device in which the fluid channels of Example 6 were not stacked was used.

Experimental example 1: Using a microfluidic device radioimmunoassay Perform

The radioactivity of the microfluidic device for radioimmunometry treated according to Examples 1, 2, 3 and 4 was measured and shown in Tables 1 and 2 below. Meanwhile, the concentrations of the T4 antigen in the liquid samples according to Examples 1, 2, 3 and 4 are shown in Table 1 below.

Radioactivity measurement result (CPM) Antigen concentration (μg/dL) Example 1 480 2 Example 2 329 4 Example 3 97 8 Example 4 433 2.6

As a result of measuring the radioactivity of the microfluidic device for radioimmunometry treated according to Examples 1 to 3, a standard calibration curve having a coefficient of determination (R ² ) of 0.9951 could be derived.

On the other hand, the concentration of the antigen present in the quality control material was inversely calculated using the standard calibration curve derived above from the radioactivity of the microfluidic device for radioimmunometry treated according to Example 4, and it was confirmed that it was 2.6 μg/dL. It is included in the appropriate concentration range (2.5 to 4.5 μg/dL) of the quality control substance presented by the radioimmunodiagnostic kit, and it is known that radioimmunoassay can be appropriately performed using the microfluidic device provided in the present invention. can

Experimental example 2: Test tube contamination evaluation by microfluidic devices

After carrying out Example 1, after removing the microfluidic device in the test tube of Example 1, the process of measuring radioactivity by relocating the test tube to the detector of the gamma count was repeated, and the isotope-labeled antigen was attached to the microfluidic device. The presence or absence of contamination by the test tube was evaluated. Specifically, according to Example 1, after measuring the radioactivity of the test tube including the microfluidic device, the radioactivity of the test tube from which the microfluidic device was removed was measured, and then the same microfluidic device was put back into the test tube and the radioactivity was measured. The process was repeated. The radioactivity measurement results according to the repeated execution of the above example are shown in FIG. 3 .

As a result of measuring the radioactivity of the test tube according to the procedure as described above, contamination of the test tube by the microfluidic device to which the isotope-labeled antigen was attached did not occur. Therefore, when performing radioimmunoassay using a microfluidic device, the test tube can be reused, unlike the conventional radioimmunoassay, and thus solid waste can be reduced.

Experimental example 3: Evaluation of protein adhesion improvement by gas injection

The radioactivity of the microfluidic device for radioimmunometry treated according to Example 5 and Comparative Example 1 was measured and shown in Table 2 and FIG. 5 below. In addition, the radioactivity compared to the amount of liquid sample required for radioimmunometry using the microfluidic device according to Example 5 and Comparative Example 1 is shown in Table 2 below.

Radioactivity measurement result (CPM) Radioactivity to Required Liquid Sample (CPM/μL) Example 5 170 19.2 Comparative Example 1 155 11.9

As a result of injecting the liquid sample into the microfluidic device according to Example 5 and Comparative Example 1, even though the same liquid injection flow rate was set, the actual liquid injection flow rate by gas injection was confirmed to be about 68%. Therefore, it was attempted to compare the radioactivity measured in Example 5 and Comparative Example 1 based on the amount of liquid sample required, and as shown in FIG. It was confirmed that it showed radioactivity. Therefore, it can be seen that the protein adhesion performance can be improved through the agitation effect on the liquid sample in the channel during radioimmunometry in a microfluidic device accompanied by gas injection.

Experimental example 4: Evaluation of amplification of detection signals by stacked structure

The radioactivity of the microfluidic device for radioimmunometry treated according to Examples 6 to 8 was measured and shown in Table 3 and FIG. 7 below. Meanwhile, the number of stacks of the microfluidic devices according to Examples 6 to 8 is shown in Table 3 below.

Radioactivity measurement result (CPM) Number of stacks (pcs) Example 6 553 3 Example 7 367 2 Example 8 180 One

As a result of measuring the radioactivity of the microfluidic device for radioimmunometry treated according to Examples 6 to 8, the laminated sheet and the detection signal showed a linearly increasing relationship, and it was confirmed that the coefficient of determination (R ² ) was 0.9999. .

Therefore, it can be seen that the detection signal can be amplified during radioimmunometry using the microfluidic device of the stacked structure provided in the present invention.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible within the scope without departing from the technical spirit of the present invention described in the claims. It will be apparent to those of ordinary skill in the art.

Claims (16)

A body having a fluidic channel attached therein to an antibody that specifically binds to an analyte labeled with a radioisotope;
the fluid channel,
a liquid inlet connected to one end of the fluid channel;
a gas inlet in communication with the fluid channel for gas introduction;
a sensing unit comprising a patterned fluid channel; and
and a fluid outlet connected to the other end of the fluid channel,
The sensing unit is disposed at one end of the microfluidic device, and the liquid inlet and the fluid outlet are disposed at the other end of the microfluidic device.
According to claim 1, wherein the liquid inlet, the sensing unit and the fluid outlet are sequentially arranged along the fluid channel, the gas inlet is a fluid channel between the liquid inlet and the sensing unit a gas input line joined to the liquid input line; Connected, microfluidic device for radioimmunometry.
The microfluidic device for radioimmunometry according to claim 1, wherein the liquid sample is introduced into the liquid inlet at a flow rate of 1 to 100 μL/min, and the gas is introduced into the gas inlet at a flow rate of 1 to 200 μL/min. .
The microfluidic device for radioimmunometry according to claim 1, wherein the ratio of the length of the liquid and gas section in the sensing unit after the input of the liquid sample and the gas is completed is 1: 0.25 to 1.5.
The microfluidic device for radioimmunometry according to claim 4, wherein the ratio of the fluid channel width to the length of the liquid section in the sensing unit is 1:1 to 1:7 after the input of the liquid sample and the gas is completed.
The method of claim 1, wherein the inside of the microfluidic channel is surface-treated with at least one residue of an amine group, a carboxyl group, a thiol group, an aldehyde group, and a hydroxyl group. An antibody-attached microfluidic device for radioimmunometry.
delete delete The microfluidic device for radioimmunometry according to claim 1, wherein the body in which the fluid channels are formed is in the form of a laminate in which two or more sheets in which the fluid channels are formed are stacked.
The microfluidic device for radioimmunometry according to claim 1, wherein the microfluidic device is for measurement by an analysis device selected from the group consisting of a gamma counter, a gamma spectrometer, and a liquid scintillation counter.
The microfluidic device for radioimmunometry according to claim 10, wherein the microfluidic device has a width of 2 to 20 mm, a length of 3 to 70 mm, and a thickness of 50 μm to 14 mm.
The microfluidic device for radioimmunometry according to claim 9, wherein the microfluidic device has a total thickness of 50 μm to 14 mm for insertion of a well type detector.
13. A method comprising: introducing a liquid sample containing an analyte labeled with a radioisotope into the liquid inlet of the microfluidic device for radioimmunometry according to any one of claims 1 to 6 and 9 to 12; and
Injecting air into the gas inlet
Including, the step of introducing the liquid sample into the liquid inlet is performed simultaneously with or alternately with the step of introducing air into the gas inlet, the radioimmunoassay method.
delete The method of claim 13, wherein the step of introducing the liquid sample into the liquid inlet and the step of introducing air into the gas inlet are performed simultaneously, wherein the liquid sample is introduced into the liquid inlet at a flow rate of 1 to 100 μL/min, The gas is injected at a flow rate of 1 to 200 μL/min, a radioimmunometry method.
The method of claim 13 , further comprising: placing the microfluidic device on a detector of an analysis device after the input of the liquid sample and the gas is completed; and
Further comprising the step of confirming the result using the gamma counter, radioimmunometry method.

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