KR102363041B1 - Lateral flow assay strip and method for quantitative analysis using thereof - Google Patents

Abstract

The present invention provides a lateral flow analysis strip using a single membrane and an analysis method using the same. The lateral flow analysis strip of the present invention is a detection particle dispersed in a sample solution and having a first binding material capable of specifically binding to a target biological material contained in the sample solution modified on the surface, extending in a first direction and extending in the first direction A porous membrane pad having a sample dropping area, a test area, and a control area spaced apart from each other and sequentially positioned along the porous membrane pad, the porous membrane pad including pores capable of moving the sample solution in the first direction using capillary action, the test A first fluorescent particle disposed on the surface of the region or inside the pores, having a diameter equal to or larger than the pore size, a second binding substance capable of specifically binding to the first binding substance is modified on the surface of the first fluorescent particle, and the control A third binding material that is disposed on the surface of the region or inside the pores, has a diameter equal to or larger than the pore size, and can specifically bind to the target biological material, and a fourth binding material that can specifically bind to the first binding material The binding material includes a second fluorescent particle modified on the surface.

Description

Lateral flow analysis strip and analysis method using the same

The present invention relates to a lateral flow analysis strip for performing quantitative analysis of a target biomaterial and an analysis method using the same, and more particularly, to a lateral flow analysis strip using a single membrane and detection particles.

Immunochromatography-based lateral flow analysis is a diagnostic method that can intuitively check whether or not a disease is infected on a membrane using an antigen-antibody reaction. while using the phenomenon that the detector is fixed to a certain part of the membrane by the antigen-antibody reaction.

The lateral flow assay strip using the conventional lateral flow analysis method includes a sample pad into which the sample is injected, a conjugation pad in which a biochemical reaction occurs between the sample and the detector, a measurement pad in which the immunoassay is performed while the sample flows by capillary action, An absorbent pad disposed downstream of the measurement pad to absorb excess sample to maintain lateral flow, and the like are arranged in one direction.

However, the black strip with this structure is complicated to manufacture because various types of pads and membranes must be used for each component, and development and production are difficult due to the consideration of the lateral flow characteristics of the membrane constituting each pad depending on the sample. There is this.

One object of the present invention is to provide a lateral flow analysis strip in which the existing lateral flow assay strip, which requires several types of pads and membranes, is implemented with a single membrane.

Another object of the present invention is to provide an analysis method using the lateral flow analysis strip.

A lateral flow analysis strip for one purpose of the present invention is a detection particle dispersed in a sample solution and a first binding material capable of specifically binding to a target biological material contained in the sample solution is modified on the surface, extending in a first direction, A porous membrane having a sample dropping region, an inspection region, and a control region that are spaced apart from each other and sequentially positioned along the first direction, and including pores capable of moving the sample solution in the first direction using capillary action First fluorescent particles disposed on the pad, the surface of the inspection area, or inside the pores, having a diameter equal to or larger than the pore size, and having a second binding material capable of specifically binding to the first binding material modified on the surface of the first fluorescent particle , and a third binding material that is disposed on the surface or inside the pores of the control region, has a diameter equal to or larger than the pore size, and is capable of specifically binding to the target biological material and to specifically bind to the first binding material A fourth binding material capable of including a second fluorescent particle modified on the surface.

In one embodiment, the detection particle may be formed of any one selected from gold, silver, copper, platinum, and palladium, or a composite of two or more.

In one embodiment, the porous membrane pad may be made of any one selected from silica, cellulose, nitrocellulose, nylon, polyethersulfone, polyvinylidene fluoride, and polytetrafluoroethylene.

In one embodiment, the pore size of the porous membrane pad may be 1 to 8㎛.

In an embodiment, the diameters of the first and second fluorescent particles may be 80% or more and 200% or less of the pore size of the porous membrane pad.

In an embodiment, the fluorescent particle may include a polymer core particle and a fluorescent nanoparticle covering the surface of the polymer core particle.

In an embodiment, the size of the polymer core particles may be 1 to 8 μm, and the size of the fluorescent nanoparticles may be 40 to 100 nm.

A method for quantitative analysis of a target biological material in a sample using the lateral flow analysis strip for another purpose of the present invention is a first binding capable of specifically binding to the target biological material in a sample containing the target biological material at an unknown concentration A first step of preparing a mixed sample by adding detection particles modified with a material to the surface, dropping the mixed sample into the sample dropping area and moving the mixed sample through a capillary phenomenon, thereby transferring the mixed sample to the first fluorescent particles in the test area and a second step of sequentially reacting with the second fluorescent particles in the control region, and measuring first and second fluorescence signals respectively generated from the first and second fluorescent particles in the inspection region and the control region and a third step of analysis.

In an embodiment, in the first step, the target biological material may be bound to the surface of the detection particle by the first binding material.

In one embodiment, in the second step, the first fluorescent particles in the inspection region are combined with the detection particles having the first binding material modified on the surface, and the second fluorescent particles in the control region are the target living body. The material and the first binding material may bind to the surface-modified detection particle.

According to the present invention, by constructing a lateral flow analysis strip using a single membrane, the process of precisely combining the pads composed of various types of conventional membranes, the process of considering the fluid movement characteristics of the sample in each pad, and each sample according to the sample Since the pad optimization process and the like can be omitted, it is possible to reduce the complexity of the production and structure of the lateral flow analysis strip.

1 is a view showing a structure for explaining a lateral flow analysis strip of the present invention.
2 and 3 are diagrams showing the structure for explaining the analysis method using the lateral flow analysis strip of the present invention.
4 is a view showing the results of cTnI quantitative analysis-1 of Example 1 of the present invention.
5 is a view showing the results of cTnI quantitative analysis-2 of Example 2 of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. Since the present invention can have various changes and can have various forms, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to the specific disclosed form, it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention. In describing each figure, like reference numerals have been used for like elements.

The terms used in the present application are only used to describe specific embodiments and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that a feature, step, operation, component, part, or combination thereof described in the specification is present, and includes one or more other features or steps. , it should be understood that it does not preclude the possibility of the existence or addition of an operation, a component, a part, or a combination thereof.

Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention 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 should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

1 is a view showing a structure for explaining a lateral flow analysis strip of the present invention.

1, the lateral flow analysis strip of the present invention is a detection particle dispersed in a sample solution and a first binding material capable of specifically binding to a target biological material contained in the sample solution is modified on the surface of the detection particle, in a first direction A sample dropping area, a test area, and a control area extending and spaced apart from each other along the first direction and having a control area, and including pores capable of moving the sample solution in the first direction using capillary action The porous membrane pad, disposed on the surface or inside the pores of the inspection area, having a diameter equal to or larger than the pore size, and a second binding material capable of specifically binding to the first binding material is modified on the surface of the first A third binding material that is disposed on the surface or inside the pores of the fluorescent particle, and the control region, has a diameter equal to or larger than the pore size, and can specifically bind to the target biomaterial, and the first binding material The fourth binding material capable of binding may include a second fluorescent particle modified on the surface.

The sample may mean a biological material suspected of containing the target biological material to be analyzed. For example, the sample may be a substance such as blood, urine, saliva, and sweat.

The target biological material means a biological material to be analyzed using the lateral flow analysis strip. For example, the biomaterial may be a material such as an antigen, an antibody, or a protein. Preferably, the biomaterial may be an antigen.

The binding material may refer to a substance that mediates binding of the detection particle and the target biological material, the detection particle and the fluorescent particle, and the fluorescent particle and the target biological material. For example, when the target biological material is an antigen, the first biological material may be an antibody that specifically binds to the antigen, and the second biological material may be an antigen that competes with the antigen; The fourth biomaterial may be an antibody that specifically binds to the antibody.

The detection particle may be modified on the surface of a first binding material capable of specifically binding to a target biological material to bind to the target biological material in the sample solution. In addition, the detection particles may be formed of a material having a quenching ability for a fluorescent material. For example, the detection particle may be formed of any one selected from gold, silver, copper, platinum, and palladium, or a composite of two or more. Preferably, the detection particle may be formed of gold. The shape of the detection particle is not particularly limited, but may be, for example, a nanorod shape.

In an embodiment, the detection particle may be a gold nanorod particle.

The porous membrane pad is capable of moving the sample within the membrane pad by capillary action, and the porous membrane pad includes silica, cellulose, nitrocellulose, nylon, polyethersulfone, polyvinylidene fluoride, and polytetra It may be formed of any one selected from fluoroethylene and the like. However, it is not necessarily limited thereto.

The pore size of the porous membrane pad may be about 1 to 8 μm. Preferably, the pore size of the porous membrane pad may be about 1 to 6 ㎛.

The diameters of the first and second fluorescent particles may be about 80% or more and 200% or less of the pore size of the porous membrane pad. When the diameters of the first and second fluorescent particles are less than about 80% of the pore size of the porous membrane pad, the first and second fluorescent particles cannot be fixed to the surface or pores of the porous membrane pad and the pores of the membrane In the case where it is difficult to escape through the gap and react with the sample, and the diameters of the first and second fluorescent particles exceed about 200% of the pore size of the porous membrane pad, in quantitative analysis using the strip, the first and second There is a problem in that the fluorescent particles are not maintained and fixed in the inspection area and the control area, but are washed away.

The fluorescent particle is a particle capable of emitting a fluorescent signal, and the fluorescent particle may include a polymer core particle and a fluorescent nanoparticle covering a surface of the polymer core particle. The polymer core and the fluorescent nanoparticles may be formed of a non-toxic material. For example, the polymer core may be formed of polystyrene, and the fluorescent nanoparticles are a TBP-Ln(III)-based complex, a DTPA-cs124-Ln(III)-based complex, which is a lanthanide complex for imparting fluorescence properties to particles. complex, BHHCT-Ln(III) series complex, BPTALn(III) series complex, BCPDA-Ln(III) series complex, and the like may be formed including any one selected from or a combination thereof.

In an embodiment, the size of the polymer core particles may be about 1 to 8 μm, and the size of the fluorescent nanoparticles may be about 40 to 100 nm.

In another embodiment, the size of the polymer core particles may be about 1.5 to 2.5 μm, and the size of the fluorescent nanoparticles may be about 70 to 80 nm.

2 and 3 are diagrams showing the structure for explaining the analysis method using the lateral flow analysis strip of the present invention.

Referring to FIG. 2 , a first step of preparing a mixed sample by adding detection particles modified on the surface of a first binding material capable of specifically binding to the target biological material to a sample containing the target biological material at an unknown concentration , a second step of sequentially reacting the mixed sample with the first fluorescent particles in the inspection region and the second fluorescent particles in the control region by dropping the mixed sample into the sample dropping region and moving it through capillary action, and the inspection region and a third step of measuring and analyzing respective first and second fluorescence signals respectively generated from the first and second fluorescent particles in the control region.

In the first step, the target biological material may be bound to the surface of the detection particle by the first binding material.

In the second step, the first fluorescent particles in the inspection region are bound to the detection particles having the first binding material modified on the surface, and the second fluorescent particles in the control region are the detection particles to which the target biomaterial is bound. The particles and the first binding material may bind to the detection particles modified on the surface.

Referring to FIG. 3, the analysis method using the lateral flow analysis strip of the present invention will be described in more detail as follows.

When analyzing a sample containing a target biological material using the lateral flow analysis strip of the present invention, the target biological material is bound to the surface of the detection particle by the first binding material during the first step A biomaterial-detecting particle complex may be formed. The mixed sample including the complex may be moved to the test area by capillary action. In the mixed sample moved to the test region, the target biomaterial is bound to the first binding substance of the detection particles, and the second binding substance specifically binding to the first binding substance is modified on the surface of the first binding substance. It may not react with the fluorescent particles and may migrate to the control region through capillary action.

On the other hand, when analyzing a sample that does not contain a target biological material, the first binding material of the detection particle of the mixed sample moved to the test region and the second binding of the first fluorescent particle in the test region They can react with substances and bond with each other. In this case, since the first fluorescent particle and the detection particle are coupled at a very close distance, the fluorescence characteristic of the first fluorescent particle may be quenched by the detection particle.

Accordingly, by measuring the first fluorescence signal in the test region, the presence or absence of a target biological material in the sample may be quantitatively analyzed. At this time, since the second fluorescent particle of the control region is designed to always capture the detection particle, it maintains the quenched state regardless of the concentration and presence of the target biomaterial, thereby ensuring the reliability of the lateral flow analysis strip of the present invention. have.

On the other hand, when the target biological material is contained in various concentrations in the sample, as the concentration of the target biological material is higher, most of the detection particles combine with the target biological material to form a complex, so that the first fluorescence in the test area Since there are few detection particles binding to the particles, the fluorescence of the first fluorescent particles cannot be quenched, so that the first fluorescence signal is measured to be high. In contrast, the lower the concentration of the target biological material in the sample, the lower the first fluorescence signal is measured, so it is possible to perform quantitative analysis of the target biological material according to the concentration by comparing the results of the first fluorescence signal. have.

According to the present invention, since the structure of the strip is simplified by using a single membrane while maintaining the same method of measuring and analyzing the results as in the conventional lateral flow strip, it can be easy to enter the bio field.

Hereinafter, a lateral flow analysis strip of the present invention and an analysis method using the same will be described in more detail through specific examples.

Purpose biomaterial

As the target biomaterial, cardiac troponin I (hereafter referred to as cTnI), which is an acute myocardial infarction diagnostic marker, was used.

Lateral Flow Analysis Strips

As the porous membrane pad, a 60 mm x 4 mm FUSION 5™ membrane having a pore size of about 2.3 μm was used, and fluorescent nanoparticles having a diameter of about 70 to 80 nm were used as fluorescent particles in the inspection and control regions of the porous membrane pad. Polystyrene particles having a diameter of about 2 μm with the particles covering the surface were used. Gold nanorods of 60 × 25 nm were used as detection particles.

Example 1: Quantitative analysis of cTnI-1

Quantitative analysis of cTnI present at various concentrations (0, 0.5, 1 ng/mL) in phosphate buffered saline (PBS) was performed using a lateral flow assay strip. A FUSION 5™ membrane was used as a measuring strip, and it was cut into a size of 60 mm X 4 mm. Fluorescent particles modified with cTnI were applied to the test area, and fluorescent particles modified with anti-mouse IgG were applied to the control area.

Here, the method for synthesizing the fluorescent particles and the detection particles used for the measurement is as follows. First, the fluorescent particles used in the inspection area and the control area are prepared in the form of modified polystyrene particles with a size of 2 μm and fluorescent nanoparticles with a size of 70 nm to 80 nm. 800 μl of 100 mM PBS solution containing 80 μg of fluorescent nanoparticles was mixed with 800 μl of 50 mM MES buffer containing 8 μg of polystyrene particles having a primary amine group on the surface, 350 μg of EDC and 595 μg of sulfo-NHS 4 The reaction was carried out at ℃ for 12 hours. After that, 100 μl of 100 mM PBS buffer containing 20 mM ethanolamine is added and reacted again at 4°C for 30 minutes. After rotating at 12000 g for 30 minutes using a centrifuge, the supernatant was separated to remove unreacted residues. Then, 1 mL of distilled water was added to make a suspension and centrifugation was performed three more times. Finally, after removing all of the supernatant generated through centrifugation, it was stored in a 100 mM PBS solution to which 1% (w/v) BSA was added.

The synthesized fluorescent particles were modified with cTnI and anti-mouse IgG in the test and control regions, respectively. First, after centrifuging the stored fluorescent particles and removing all the supernatant, 1 mL of 100 mM PBS solution containing 5 mg/mL of BS ³ is added, redispersed, and reacted at 4° C. for 30 minutes. After rotating at 12000 g for 30 minutes using a centrifuge, the supernatant was separated to remove unreacted residues. Thereafter, 1 mL of 100 mM PBS buffer was added to make a suspension, and the centrifugation process was performed three more times. After removing all the supernatant, 800 μl of 100 mM PBS solution containing 10 μg/mL of cTnI or anti-mouse IgG is added, the particles are redispersed, and the reaction is carried out at 4° C. for 1 hour. Thereafter, 200 μl of 100 mM PBS containing 20 mM ethanolamine is added and further reacted at 4° C. for 30 minutes. After rotating at 12000 g for 30 minutes using a centrifuge, the supernatant was separated to remove unreacted residues. Then, 1 mL of 100 mM PBS was added to make a suspension and centrifugation was performed three more times. After centrifugation, all of the supernatant was removed and re-dispersed in 800 μl of lining buffer and stored.

The prepared fluorescent particles were applied to the inspection and control regions of the membrane, respectively, and the membrane was vacuum-dried for 2 hours after application of the particles.

The detection particles were prepared by modifying a 60 nm X 25 nm gold nanorod with 16A11 monoclonal antibody, an antibody against cTnI. Antibody formulation was followed by the method provided by cytodiagnostic, a manufacturer of gold nanorods.

Quantitative analysis of cTnI proceeds in the following manner. First, in order to confirm the fluorescence signal before fluorescence quenching occurs, the fluorescence signal of the prepared strip is obtained. The fluorescence signal is obtained by irradiating excitation light for 300 μsec, removing the excitation light, and summing the signals between 100 μsec and 700 μsec. Thereafter, in order to confirm the degree of extinction of fluorescence according to the concentration of cTnI, the following procedure is performed. First, the detection particles are diluted to a concentration of OD 1 in a 100 mM PBS solution containing 1% (w/v) BSA, mixed with a sample containing cTnI, and reacted at room temperature for 20 minutes. After completion of the reaction, the mixed sample is dropped at the beginning of the strip, and then 100 μl of PBS buffer is added dropwise to the same area. After the solution is advanced to the end of the strip using capillary action, a fluorescence signal is obtained. When the residual fluorescence signal is converted into % by comparing the fluorescence signal after the drop of the sample with the conventional fluorescence signal, it can be confirmed that the higher the concentration of cTnI, the higher the remaining fluorescence signal is measured.

The fluorescence signal measured through the lateral flow analysis strip is shown in FIG. 4 .

Referring to FIG. 4 , a difference in the first fluorescence signal measured in the test region according to the concentration of cTnI may be confirmed. It can be seen that the higher the concentration of cTnI, the higher the first fluorescence signal was measured.

Example 2: Quantitative analysis of cTnI-2

Example 2 proceeds in the same manner as in Example 1, and the solution used as the base of the sample was changed from 100 mM PBS to cTnI free serum, and conditions similar to those of the actual plasma sample were applied.

Quantitative analysis of cTnI present in human serum at various concentrations (0, 0.15, 0.29, 0.58, 1.16 ng/mL) was performed using a lateral flow assay strip. The fluorescence signal measured through the lateral flow analysis strip is shown in FIG. 5 .

Referring to FIG. 5 , it can be seen that the detection limit is about 0.097 ng/mL.

Although the above has been described with reference to the preferred embodiments of the present invention, those skilled in the art can variously modify and change the present invention within the scope without departing from the spirit and scope of the present invention as set forth in the following claims. You will understand that you can.

1: Porous Membrane Pad
2: Sample loading area
3: Inspection area
4: Contrast area
A: sample
A-1: Target biological material
A-2: Detection Particles
3-1: first fluorescent particle
4-1: second fluorescent particle
5-1: detection particle combined with the first fluorescent particle in the inspection area
5-2: Detecting particles bound to the target biological material of the sample
5-3: detection particle combined with the second fluorescent particle in the control region

Claims (10)

a detection particle dispersed in the sample solution and having a first binding material modified on the surface of the first binding material capable of specifically binding to a target biological material contained in the sample solution;
A sample dropping area, an inspection area and a control area extending in a first direction and spaced apart from each other along the first direction and having a control area, the sample solution can be moved in the first direction using a capillary phenomenon a porous membrane pad comprising pores;
a first fluorescent particle disposed on the surface of the inspection area or inside the pores, having a diameter equal to or greater than the pore size, and having a second binding material capable of specifically binding to the first binding material modified on the surface thereof; and
A third binding material that is disposed on the surface or inside the pores of the control region, has a diameter equal to or larger than the pore size, and can specifically bind to the target biological material, and a third binding material capable of specifically binding to the first binding material The lateral flow analysis strip, wherein the fourth binding material comprises a second fluorescent particle modified on the surface.
The method of claim 1,
The detection particle is characterized in that it is formed of any one selected from gold, silver, copper, platinum and palladium,
Lateral flow analysis strips.
The method of claim 1,
The porous membrane pad is formed of any one selected from silica, cellulose, nitrocellulose, nylon, polyethersulfone, polyvinylidene fluoride and polytetrafluoroethylene,
Lateral flow analysis strips.
The method of claim 1,
The pore size of the porous membrane pad is 1 to 8 μm,
Lateral flow analysis strips.
5. The method of claim 4,
The diameter of the first and second fluorescent particles is characterized in that 80% or more and 200% or less of the pore size of the porous membrane pad,
Lateral flow analysis strips.
6. The method of claim 5,
The fluorescent particles are
Containing a polymer core particle and fluorescent nanoparticles covering the surface of the polymer core particle,
Lateral flow analysis strips.
7. The method of claim 6,
The size of the polymer core particles is 1 to 8 μm,
The size of the fluorescent nanoparticles is 40 to 100 nm,
Lateral flow analysis strips.
In the quantitative analysis method of a target biological material in a sample using the lateral flow analysis strip according to claim 1,
a first step of preparing a mixed sample by adding detection particles modified on the surface of a first binding material capable of specifically binding to the target biological material to a sample containing the target biological material at an unknown concentration;
a second step of sequentially reacting the mixed sample with the first fluorescent particles of the test area and the second fluorescent particles of the control area by dropping the mixed sample into the sample dropping area and moving it through capillary action; and
and a third step of measuring and analyzing respective first and second fluorescence signals generated from the first and second fluorescent particles in the test region and the control region, respectively.
9. The method of claim 8,
In the first step, the target biological material is characterized in that the binding to the surface of the detection particle by the first binding material,
A lateral flow analysis strip analysis method.
9. The method of claim 8,
In the second step,
The first fluorescent particles in the inspection area are combined with the detection particles on which the first binding material is modified,
The second fluorescent particle of the control region is characterized in that the target biological material and the first binding material are combined with the detection particle modified on the surface,
A lateral flow analysis strip analysis method.

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