Lateral flow membrane sensor capable of detecting scattered light

Abstract

The present invention relates to a lateral flow membrane sensor and, specifically, to a membrane sensor in which the detection sensitivity for a sample is enhanced by using a scattered light signal from a marker.

Description

Lateral flow membrane sensor capable of detecting scattered light

[0001] The present invention relates to a lateral flow membrane sensor, and more particularly, to a membrane sensor that reduces background scattered light signals and effectively detects scattered light signals of a labeling substance, thereby improving the detection sensitivity of a sample.

[0002] A biosensor is an analytical device that combines a bioreceptor with signal transduction technology to selectively measure and analyze specific substances at the molecular level. Bioreceptors, such as antibodies, enzymes, aptamers, and cells, selectively recognize the substance of interest. Signal transduction technology detects the changes that occur when the bioreceptor reacts or binds with the target substance, converting these changes into a signal that can be recognized by humans.

[0003] Biosensors are being used in a wide range of fields, including medicine, food, the environment, and the military. They are particularly useful for disease and infection detection in hospitals. They are also being used most frequently for self-diagnosis of blood sugar levels at home.

[0004] Lateral flow assays (LFA) are immunoassays that can be used to detect various analytes in biological samples. A common LFA method uses capture antibodies immobilized at specific locations on a nitrocellulose membrane, for example. The advantage of LFA, unlike ELISA, is that the membrane allows for a single-step analysis. Based on the principles of high affinity, sensitivity, and selectivity between specific antibody-antigen pairs, immunologically-based assays are more widely applicable due to the wide variety of existing antibodies and the availability of affordable reaction reagents. Lateral flow technology is highly suitable for point-of-care disease diagnosis because it is reliable and inexpensive, without requiring power, cold chain storage and transport, or specialized reagents.

[0005] Conventional LFA-based membrane sensors have limitations in measuring the scattered light signal of a labeling substance because the scattered light signal (background signal) generated from the membrane, which is the reaction zone, is greater than the scattered light signal of the labeling substance.

[0006] [Prior Art Literature]

[0007] Korean Patent Publication No. 2013-0037648

[0008] The present invention provides a membrane sensor that improves the signal intensity of a detection target in a sample, and relates to a membrane sensor capable of measuring the scattered light signal of a label material by minimizing the scattered light signal of the reaction pad by making the reaction pad transparent.

[0009] In addition, the technical problems to be solved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0010] In order to achieve the above object, a lateral flow membrane sensor according to one embodiment of the present invention includes a sample pad into which a sample containing a reaction solution is injected; a reaction pad on which the sample is spread and on which optical characteristics are controlled by the reaction solution; and a light absorption pad disposed below the reaction pad and absorbing light irradiated onto the reaction pad.

[0011] It may be to detect the signal of scattered light of the label material in the above reaction pad.

[0012] The above-mentioned labeling material may be a nanoparticle, and the nanoparticle may have scattering properties with respect to incident light.

[0013] The above reaction pad may absorb the reaction solution and transmit light.

[0014] The above reaction pad may be determined by the refractive index of the reaction solution, and the difference between the refractive index of the reaction pad and the refractive index of the reaction solution may be 0.12 or less.

[0015] The above light-absorbing pad may have a light reflectivity lower than that of barium sulfate (BaSO4), and the above light-absorbing pad may have a light reflectivity of 13% or less of that of barium sulfate (BaSO4).

[0016] The present invention has the effect of limiting scattered light from a reaction pad for a lateral flow membrane sensor, improving the intensity of a detection signal of a sample by using scattered light from a labeling material, and thus improving detection sensitivity even at low concentrations of the sample.

[0017] FIG. 1a and FIG. 1b illustrate a configuration diagram of a lateral flow membrane sensor according to one embodiment of the present invention.

[0018] FIG. 2 illustrates the optical signal detection principle of a lateral flow membrane sensor according to one embodiment of the present invention.

[0019] FIG. 3 illustrates the diffuse reflectance (scattered light) and absorbance of a reaction pad according to the refractive index of a reaction solution of a fluid membrane sensor according to one embodiment of the present invention.

[0020] FIG. 4 illustrates a detection signal according to the refractive index of a reaction solution of a fluid membrane sensor according to one embodiment of the present invention.

[0021] FIG. 5a and FIG. 5b illustrate detection signals according to the reflectivity of a light-absorbing pad of a fluid membrane sensor according to one embodiment of the present invention.

[0022] FIG. 6 illustrates a detection signal according to light exposure time of a fluid membrane sensor according to one embodiment of the present invention.

[0023] Figure 7 illustrates the results of a detection concentration analysis of a fluid membrane sensor according to one embodiment of the present invention.

[0024] Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement them. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[0025]

[0026] Referring to FIG. 1A below, in one embodiment of the present invention, a lateral flow membrane sensor (100) includes a sample pad (110); a conjugate pad (120); a reaction pad (130); a light absorption pad (140); and an absorption pad (150).

[0027]

[0028] More specifically, a lateral flow membrane sensor (100) according to one embodiment of the present invention includes a sample pad (110) in accordance with the flow direction of a sample; a conjugate pad (120) in contact with the sample pad (110); a reaction pad (130) in contact with the conjugate pad (120); a light absorption pad (140) disposed below the reaction pad; and an absorption pad (150) in contact with the reaction pad (130).

[0029] The sample pad (110) is an area where a sample is loaded, and performs the function of uniformly distributing the sample and spreading the sample onto the reaction pad (120).

[0030] The sample pad (110) is in contact with the conjugate pad (120), and the type of the sample pad (110) is not limited as long as it is a material capable of absorbing a liquid sample, and preferably may be cellulose, polyester, polypropylene, or glass fiber.

[0031] The sample is not particularly limited as long as it can contain the specimen to be detected. For example, the sample may be a biological sample, such as a biological fluid or biological tissue. Examples of biological fluids include urine, blood (whole blood), plasma, serum, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, and amniotic fluid. A biological tissue is an aggregate of cells, and generally includes intracellular substances that form one of the structural materials of a human, animal, plant, bacterial, fungal, or viral structure, and a specific type of aggregate, such as connective tissue, epithelial tissue, muscle tissue, and nervous tissue. Examples of biological tissues may also include organs, tumors, lymph nodes, arteries, and individual cell(s).

[0032] An analyte may be understood to mean a molecule or other substance within a sample to be detected. For example, an analyte may include an antigenic substance, a ligand (mono- or polyepitope), a hapten, an antibody, or a combination thereof. Specifically, an analyte may include, but is not necessarily limited to, toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, drugs, drug intermediates or byproducts, bacteria, viral particles, yeasts, fungi, protozoa, and metabolites or antibodies to the foregoing substances. However, it may typically be an antigen or an antibody.

[0033] The above sample may further include a reaction solution, and the reaction solution may be capable of transmitting light incident on the reaction pad (130) by lowering the light reflectance of the reaction pad (130) after being deployed on the reaction pad (130). The reaction solution may include a surfactant, a polymer, and a polar organic solvent, and the surfactant may be Triton X-100 (C 14 H 22O(C2H4O) n (n=9-10))(refractive index (n) = 1.49), the polymer may be PVA (poly(vinyl alcohol))(refractive index (n) = 1.47), and the polar organic solvent may be dimethyl sulfoxide (refractive index (n) = 1.47).

[0034]

[0035] The conjugate pad (120) may be placed between the sample pad (110) and the membrane pad (130).

[0036] The conjugate pad (120) may include a labeling material that generates a signal detectable by the naked eye or using a sensor. The labeling material may be a nanoparticle-detection antibody conjugate in which a nanoparticle and a detection antibody that binds to an antigen are linked.

[0037] The nanoparticles of the above conjugate refer to nanoparticles that function as detectable labels. The nanoparticles may have light-scattering properties with respect to incident light. The nanoparticles may most preferably be gold (Au) nanoparticles. As an example, an antigen injected into a sample can be detected at a test line through a primary assay using gold nanoparticles (Au nanoparticles) conjugated with an antibody.

[0038] The detection antibody of the above conjugate refers to an antibody that specifically binds to the antigen to be analyzed, and includes antibody fragments as long as they possess binding specificity. The detection antibody may be a monoclonal antibody or a polyclonal antibody, and a monoclonal antibody is most preferably used. The binding between the nanoparticle and the detection antibody includes, but is not limited to, ionic bonds, covalent bonds, metal bonds, coordination bonds, hydrogen bonds, and van der Waals bonds.

[0039]

[0040] The reaction pad (130) has a porous structure that allows the sample to move by capillary action. The reaction pad (130) may be a membrane. As an example, the membrane may be made of one or more selected from nitro cellulose, nylon, polysulfone, polyethersulfone, and polyvinylidene fluoride (PVDF).

[0041] The reaction pad (130) includes a detection area and a control area, and the detection area and the control area are spaced apart from each other and placed on the membrane. The detection area may be a test line (T), and the control area may be a control line (C).

[0042] The above test line is positioned before the control line based on the direction of movement (Flow) of the sample, and more specifically, may be positioned on the reaction pad (130) in the order of the test line and the control line based on the direction of movement (Flow) of the sample. As an example, the test line may be immobilized with an antibody that specifically binds to the antigen to be detected.

[0043] The above control line is positioned at the rear end of the test line based on the direction of movement of the sample, and is positioned toward the absorption pad (150).

[0044] The reaction pad (130) may be made transparent by the reaction solution contained in the sample due to its porous structure, and the reaction pad (130) may be made of a material having a refractive index of 0.12 or less of the reaction solution. The reaction pad (130) is made transparent by the reaction solution, and may transmit light when exposed to light. Since the reaction pad (130) transmits light, only the light scattered by the metal nanoparticles (the metal nanoparticles of the conjugate pad (130) bind to the antibodies of the test line) in the reaction pad (130) can be measured, and thus more sensitive detection can be possible.

[0045] More specifically, referring to FIG. 2, (a) of FIG. 2 is a conventional membrane sensor, in which a membrane pad within a conventional membrane sensor reflects incident light, and as a result, the metal nanoparticles of a test line absorb and scatter light, and as a result, the intensity of light at the test line can only be measured with a weakened intensity.

[0046] However, (b) of FIG. 2 is a lateral flow sensor (100) of the present invention, and since the reaction pad (130) transmits all incident light, only scattered light by metal nanoparticles of the test line can be measured, and as a result, more sensitive detection can be achieved along with measurement of the intensity of strong light at the test line.

[0047]

[0048] The light absorption pad (140) may absorb light passing through the reaction pad (130), and the light absorption pad (140) may be a material having low light reflectivity. For example, the light reflectivity of the light absorption pad (140) may be 13% or less of the light reflectivity of barium sulfate (BaSO4). Exemplary materials for the light absorption pad (140) may include nitrocellulose (n=1.51), cellulose acetate (n=1.47), polyvinylidene fluoride (n=1.42), or polytetrafluoroethylene (n=1.38).

[0049] Referring to FIG. 1B, the light absorption pad (141) may have a predetermined length and may be a support supporting a unit configuration of the lateral flow membrane sensor (100). More specifically, the lateral flow membrane sensor (100) may have a sample pad (110); a conjugate pad (120); a reaction pad (130); and an absorption pad (150) sequentially arranged on the light absorption pad (141).

[0050] The absorption pad (150) plays a role in absorbing a sample that has been developed into a reaction pad (130), and specifically, it plays a role in absorbing a sample that has moved through the sample pad (110), conjugate pad (120), and reaction pad (130), and providing a driving force for the sample to move through a capillary phenomenon.

[0051] The absorbent pad (150) is not limited in type as long as it is a material capable of absorbing a liquid sample, and may preferably be cellulose, polyester, polypropylene, or glass fiber.

[0052]

[0053] Hereinafter, the present invention will be described in more detail through examples. These examples are provided solely to facilitate understanding of the present invention and do not limit the scope of the present invention.

[0054]

[0055] Manufacturing Example 1. Manufacturing of a lateral flow membrane sensor

[0056] A light-absorbing pad is used as a support, and a PVC substrate having a size of 4 cm in length X 3.8 cm in width is prepared by bar-coating CNT paste on the upper surface of the PVC substrate. Then, as shown in Fig. 1, a sample pad (glass fiber; Ahlstrom), a conjugate pad (glass fiber; Ahlstrom), a reaction pad (nitrocellulose membrane; millipore), and an absorption pad (ap22; Ahlstrom), all of which are 3.8 cm in width, are sequentially placed on the prepared support to prepare a membrane sensor.

[0057]

[0058] The conjugate pad immobilized gold nanoparticles (AuNPs, 100 nm), which are chromogenic particles, by combining them with antibodies that specifically bind to the Influenza A nucleocapsid protein.

[0059] An antibody that specifically binds to the Influenza A nucleocapsid protein was dispensed onto the test line on the reaction pad and then immobilized by drying at 37°C for 15 minutes.

[0060]

[0061] Comparative Example 1.

[0062] A lateral flow membrane sensor was manufactured in the same manner as in Manufacturing Example 1 above, but without a light absorption pad.

[0063]

[0064] Example analysis.

[0065] The reflectance analysis in the experimental example described below was performed after injecting a solution containing 3,3′ (TDE, n=1.508) into the sample pad and allowing time for the solution to sufficiently spread to the reaction pad and for the antigen to be detected at the test line.

[0066]

[0067] Experimental Example 1. Analysis of detection signals according to the refractive index of the reaction solution.

[0068] When the reaction pad having a refractive index of 1.52 (nitrocellulose) was exposed to light (exposure time: 1 s) according to the concentration (1 OD (optical density), 0.1 OD, 0.01 OD) of the labeling material (gold nanoparticles conjugated with antibodies) of the lateral flow membrane sensor of the above manufacturing example 1, the diffuse relflectance and absorbance according to the refractive index of the reaction solution were measured and shown in Fig. 3, and the photographic image is shown in Fig. 4.

[0069] Referring to Fig. 3, it can be confirmed that as the refractive index of the reaction solution approaches the refractive index of the reaction pad (1.52), the diffuse reflectivity (Fig. 3 (a)) and absorbance (Fig. 3 (b)) of the reaction pad decrease, which results in an increase in the light transmittance of the reaction pad.

[0070] Referring to Fig. 4, when the refractive index of the reaction solution is adjusted to match the refractive index of the material of the reaction pad, light reflection and scattering from the reaction pad weaken, resulting in a darker background image. When the refractive index of the reaction pad and the reaction solution is within 0.12 (1.408), the light signals of the test line and the reaction pad are reversed, resulting in the light signal of the test line appearing brighter.

[0071]

[0072] Experimental Example 2. Analysis of Detection Signals According to the Reflectance of the Light Absorbing Pad

[0073] For the lateral flow membrane sensor of the above Manufacturing Example 1, when the refractive index of the reaction solution (n = 1.52) matches the refractive index of the reaction pad (n = 1.52) according to the concentration of the labeling substance (1 OD, 0.1 OD, 0.01 OD), the light detection signal photograph images when exposed to light (exposure time: 1 s, 5 s) for the reflectance of the light-absorbing pad compared to BaSO4 (compared to the light reflected by barium sulfate) are shown in FIGS. 5a and 5b.

[0074] Referring to FIGS. 5a and 5b, the lower the light reflectivity of the light-absorbing pad, the lower the scattering and reflection signals generated from the reaction pad. When the reflectivity of the light-absorbing pad becomes 13% or less compared to the reflectivity of barium sulfate, the light signals of the reaction pad and the test line are reversed, so that the signal of the test line becomes brighter. This means that when the refractive index of the reaction pad and the refractive index of the reaction solution are identical, the reaction pad becomes transparent, and thus, the light-absorbing pad can be observed with the naked eye.

[0075]

[0076] Experimental Example 3. Analysis of Detection Signals According to Exposure Time

[0077] For the lateral flow membrane sensor of the above Manufacturing Example 1, when the refractive index of the reaction solution (n = 1.52) matches the refractive index of the reaction pad (n = 1.52) and the light-absorbing pad has a low reflectance compared to the BaSO4 reflectance, a photographic image of the detection signal of light versus exposure to light is shown in Fig. 6.

[0078] Referring to Fig. 6, since only the scattering signal of the metal nanoparticles can be measured, the light exposure time can be increased during measurement to maximize the scattering signal of the metal nanoparticles.

[0079]

[0080] Experimental Example 4. Detection Concentration Analysis

[0081] For the lateral flow membrane sensor of the above Manufacturing Example 1 and Comparative Example 1, when the refractive index of the reaction solution (n = 1.52) matches the refractive index of the reaction pad (n = 1.52) and the light absorption pad has a low reflectance (1%) compared to BaSO4, when exposed to light (exposure time: 1 s), the detection signal of light compared to the detection of gold nanoparticles is shown in Fig. 7.

[0082] Referring to Figure 7, it is confirmed that Comparative Example 1 of the existing LFA method detects up to a concentration of 0.20 ng / ml, while Manufacturing Example 1 detects up to a concentration of 0.003 ng / ml.

[0083]

[0084] Through Experimental Examples 1 to 4, it can be confirmed that when the refractive index of the reaction pad material and the refractive index of the reaction solution match, the reaction pad becomes transparent, and when the reaction pad becomes transparent, the light-absorbing pad can be observed with the naked eye. At this time, it can be confirmed that the lower the light reflectance of the light-absorbing pad, the more the scattering signal of the gold nanoparticles (AuNP) is emphasized compared to the background (light-absorbing pad), thereby increasing the detection rate.

[0085]

[0086] Although the preferred 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 improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

Claims

1. Sample pad into which a sample containing a reaction solution is injected; A reaction pad on which the sample is developed and the optical properties are controlled by the reaction solution; A light-absorbing pad disposed below the reaction pad and absorbing light irradiated onto the reaction pad, Lateral flow membrane sensor.

2. In paragraph 1, Detecting the signal of scattered light of the label material in the above reaction pad, Lateral flow membrane sensor.

3. In paragraph 2, The above-mentioned labeling material is a nanoparticle, Lateral flow membrane sensor.

4. In paragraph 3, The above nanoparticles have scattering properties for incident light. Lateral flow membrane sensor.

5. In paragraph 1, The above reaction pad absorbs the reaction solution and transmits light. Lateral flow membrane sensor.

6. In paragraph 1, The above reaction pad is determined by the refractive index of the reaction solution. Lateral flow membrane sensor.

7. In paragraph 6, The difference between the refractive index of the reaction pad and the refractive index of the reaction solution is 0.12 or less. Lateral flow membrane sensor.

8. In paragraph 1, The above light absorbing pad has a light reflectance lower than that of barium sulfate (BaSO4). Lateral flow membrane sensor.

9. In paragraph 8, The above light absorbing pad has a light reflectivity of 13% or less of the light reflectivity of barium sulfate (BaSO4). Lateral flow membrane sensor.

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