Lateral flow membrane sensor

Abstract

The present invention relates to a lateral flow membrane sensor and, more particularly, to a membrane sensor that enhances the detection signal intensity of a sample by releasing TMB. The present invention was supported by a "Bio-IoT Sensor and Component Technology Upgrade Support Project of the Regional Specialization Industry Development" of Jeollanam Province. This work was supported by the Ministry of Trade, Industry and Energy (MOTIE), and Korea Institute for Advancement of Technology (KIAT) through the International Cooperative R&D Program (P0019785). Financial support for this study was provided by the National Research Foundation (NRF) grant funded by the Ministry of Science and ICT (MSIT) of Korea (2022K1A4A8A01080317).

Description

Lateral flow membrane sensor

[0001] The present invention relates to a lateral flow membrane sensor, and more particularly, to a membrane sensor that enhances the detection signal intensity of a sample by releasing TMB.

[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] (Prior art document) Korean Patent Publication No. 2013-0037648

[0006] The present invention provides a membrane sensor that improves the signal intensity of a detection target in a sample.

[0007] 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.

[0008] In order to achieve the above object, a lateral flow membrane sensor according to one embodiment of the present invention includes a sample pad; an absorbent pad; a membrane pad disposed between the sample pad and the absorbent pad; a conjugate pad disposed between the sample pad and the membrane pad; a TMB pad disposed between the membrane pad and the conjugate pad; and a TMB reinforced pad disposed on the sample pad.

[0009] The test line of the above membrane may comprise an antibody that specifically binds to any one of COVID19 (CoV), AdV (Adnovirus), Influenza A, and Influenza B.

[0010] The above conjugate pad may include platinum nanoparticles (Pt NPs).

[0011] The above TMB reinforcement pad may be shorter in length than the above sample pad.

[0012] The above TMB reinforcement pad may be placed on one side of the sample pad.

[0013] The above TMB reinforcing pad may include any one of citric acid, malic acid, maleic acid, and fumaric acid as an acid substance, and the concentration of the acid substance may be 0.01 to 0.5 M.

[0014] The above TMB reinforcing pad may include Na2O2, and the concentration of the Na2O2 may be 10 to 100 mM.

[0015] The present invention has the effect of first forming a complex of a detection substance contained in a sample and a chromogenic labeling substance present in a conjugate pad for a lateral flow membrane sensor, and then secondarily forming a chemical reaction that enhances the color development of the chromogenic labeling substance, thereby improving the intensity of a detection signal.

[0016] FIG. 1 illustrates a configuration diagram of a lateral flow membrane sensor according to one embodiment of the present invention.

[0017] FIG. 2 illustrates a schematic diagram of a lateral flow membrane sensor according to one embodiment of the present invention.

[0018] Figure 3 is a schematic diagram illustrating the operation of a lateral flow membrane sensor according to one embodiment of the present invention.

[0019] FIG. 4 illustrates detection results over time after sample injection of a lateral flow membrane sensor according to one embodiment of the present invention.

[0020] FIG. 5 illustrates detection results according to the TMB pad and TMB reinforced pad structures of a lateral flow membrane sensor according to one embodiment of the present invention.

[0021] FIG. 6 illustrates detection results according to a combination of a sample pad and an absorption pad of a lateral flow membrane sensor according to one embodiment of the present invention.

[0022] Figure 7 illustrates detection results according to the type of TMB pad of a lateral flow membrane sensor according to one embodiment of the present invention.

[0023] Figure 8 illustrates the detection results according to the acid concentration of a lateral flow membrane sensor according to one embodiment of the present invention.

[0024] Figure 9 illustrates the detection results according to the type of acid of the lateral flow membrane sensor according to one embodiment of the present invention.

[0025] Figure 10 illustrates the detection results according to the concentration of Na2O2 of a lateral flow membrane sensor according to one embodiment of the present invention.

[0026] Figure 11 illustrates the detection results according to detection temperature conditions of a lateral flow membrane sensor according to one embodiment of the present invention.

[0027] Figure 12 illustrates the time-dependent detection results of a lateral flow membrane sensor according to one embodiment of the present invention.

[0028] Figure 13a illustrates the antigen-specific signal detection results of a lateral flow membrane sensor according to one embodiment of the present invention.

[0029] Figure 13b illustrates the quantitative results of antigen-specific signal detection of a lateral flow membrane sensor according to one embodiment of the present invention.

[0030] Figure 14 illustrates the results of a multi-antigen detection experiment of a lateral flow membrane sensor according to one embodiment of the present invention.

[0031] A lateral flow membrane sensor according to one embodiment of the present invention comprises: a sample pad; an absorbent pad; a membrane pad disposed between the sample pad and the absorbent pad; a conjugate pad disposed between the sample pad and the membrane pad; a TMB pad disposed between the membrane pad and the conjugate pad; and a TMB reinforced pad disposed on the sample pad.

[0032] The test line of the above membrane may comprise an antibody that specifically binds to any one of COVID19 (CoV), AdV (Adnovirus), Influenza A, and Influenza B.

[0033] The above conjugate pad may include platinum nanoparticles (Pt NPs).

[0034] The above TMB reinforcement pad may be shorter in length than the above sample pad.

[0035] The above TMB reinforcement pad may be placed on one side of the sample pad.

[0036] The above TMB reinforcing pad may include any one of citric acid, malic acid, maleic acid, and fumaric acid as an acid substance, and the concentration of the acid substance may be 0.01 to 0.5 M.

[0037] The above TMB reinforcing pad may include Na2O2, and the concentration of the Na2O2 may be 10 to 100 mM.

[0038] 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.

[0039]

[0040] Referring to FIGS. 1 and 2 below, a lateral flow membrane sensor (100) according to one embodiment of the present invention includes a support (110); a sample pad (120); an absorption pad (130); a membrane pad (140); a conjugate pad (150); a TMB pad (160); and a TMB reinforcement pad (170).

[0041]

[0042] More specifically, a lateral flow membrane sensor (100) according to one embodiment of the present invention includes a support (110) of a length member; a sample pad (120) disposed on the support (110); an absorption pad (130) disposed on the support (110); a membrane pad (140) disposed on the support (110) and between the sample pad (120) and the absorption pad (130); a conjugate pad (150) disposed between the sample pad (120) and the membrane pad (140); a TMB pad (160) disposed between the membrane pad (140) and the conjugate pad (150), and a TMB reinforcement pad (170) disposed on the sample pad.

[0043]

[0044] The support (110) may be a longitudinal member having a strip shape, and may be configured to have a plurality of unit configurations that implement a multi-diagnosis membrane sensor (100). The support (110) may be configured to fix and support a sample pad (120); an absorption pad (130); a membrane pad (140); a conjugated pad (150); a TMB pad (160); and a TMB engagement pad (170).

[0045] The support (110) may be formed of any material that can support and transport the sample pad (120); the absorption pad (130); the membrane pad (140); the conjugate pad (150); the TMB pad (160); and the TMB reinforcement pad (170). However, it is generally preferred that the support be liquid-impermeable so that the fluid of the sample diffusing through the reaction membrane (the membrane pad (140)) does not leak through the support (110). The support (110) may be made of a glass material or a polymer material, and examples thereof include, but are not limited to, polystyrene, polypropylene, polyester, polybutadiene, polyvinyl chloride, polyamide, polycarbonate, epoxide, methacrylate, and polymelamine.

[0046] The sample pad (120) is an area where a sample is loaded, and performs the function of uniformly distributing the sample and spreading the sample onto the reaction membrane (membrane pad (140)).

[0047] The sample pad (120) is in contact with the conjugate pad (150), and the type of the sample pad (120) is not limited as long as it is a material capable of absorbing a liquid sample, and preferably, it may be cellulose, polyester, polypropylene, or glass fiber.

[0048] 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).

[0049] 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.

[0050] The absorption pad (130) plays a role of absorbing a sample developed on a reaction membrane (membrane pad (140)), and specifically, it plays a role of absorbing a sample that has moved through the sample pad (110), conjugate pad (150); TMB oxidation pad (170) and membrane pad (test line and control line) (140), and providing a driving force for the sample to move through capillary action.

[0051] The absorbent pad (130) 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] The membrane pad (140) enables the sample to move by capillary action, and the membrane pad (140) may be made of one or more selected from nitro cellulose, nylon, polysulfone, polyethersulfone, and polyvinylidene fluoride (PVDF).

[0054] The membrane pad (140) 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) (141), and the control area may be a control line (C) (142).

[0055] The test line (141) is arranged in front of the control line (142) based on the movement direction (Flow) of the sample. More specifically, the test line (141) and the control line (142) may be arranged on the membrane pad (140) based on the movement direction (Flow) of the sample.

[0056] As an example, the test line (141) may be immobilized with an antibody that specifically binds to any one of COVID19, AdV (Adnovirus), RSV, Influenza A, and Influenza B.

[0057] The control line (142) is positioned at the rear end of the test line (142) based on the direction of movement of the sample, and is positioned toward the absorption pad (130).

[0058]

[0059] The conjugate pad (150) may be placed between the sample pad (120) and the membrane pad (140), and more specifically, the conjugate pad (150) may be placed between the sample pad and the TMB oxide pad (170).

[0060] The conjugate pad (150) 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.

[0061] The nanoparticles of the above conjugate refer to nanoparticles that function as detectable labels. The nanoparticles are preferably nanoparticles of metal, and examples of the metals include, but are not limited to, noble metals such as gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iridium (Ir), rhodium (Rh), and ruthenium (Ru); transition metals such as titanium (Ti), zirconium (Zr), hafnium (Hf), vanadium (V), niobium (Nb), tantalum (Ta), chromium (Cr), molybdenum (Mo), tungsten (W), ruthenium (Ru), and osmium (Os); metals such as iron (Fe), nickel (Ni), and cobalt (Co); and metal oxides such as magnesium oxide (MgO), titanium dioxide (TiO2), vanadium pentoxide (V2O5), and zinc oxide (ZnO). The above nanoparticles may most preferably be platinum (Pt) nanoparticles or gold (Au) nanoparticles. As an example, referring to Fig. 3, an antigen injected into a sample can be detected at a test line through a primary assay using platinum nanoparticles (Pt Nanoparticles; PtNPs) conjugated with an antibody.

[0062] 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.

[0063] The TMB pad (160) may be placed on the conjugate pad (150) and the membrane pad (140), and may oxidize TMB (3,3',5,5'-Tetramethylbenzidine) by an acidic substance released from the TMB strengthening pad (170) to release ox-TMB, thereby enhancing the color development of the labeling substance captured on the test line (141) and the control line (142).

[0064] The TMB reinforcing pad (170) may be placed on the sample pad (120), and more specifically, may be placed at one end of the sample pad (120) based on the movement direction (Flow) of the sample, and more specifically, the TMB reinforcing pad (170) may be placed in the length direction from one end of the sample pad (120). The TMB reinforcing pad (170) may have a smaller area than the sample pad (120) and may have a shorter length in the movement direction (Flow) of the sample with respect to the target pad (120).

[0065] The TMB reinforcement pad (170) may include any one of citric acid, malic acid, maleic acid, and fumaric acid as an acid substance, and preferably, the acid substance may be citric acid, and the concentration of the acid substance may be 0.01 to 0.5 M. As an example, referring to FIG. 3, after a sample is injected onto the sample pad (120), citric acid at a pH of 4.0 is released from the TMB reinforcement pad (170) in about 3 to 10 minutes, and then TMB may be released onto the TMB pad (160) to perform a reinforcement reaction.

[0066] The TMB reinforcement pad (170) may include Na2O2, and the concentration of the Na2O2 may be 10 to 100 mM. The Na2O2 may be in a solid state, which can solve the problem of the activity of the reagent decreasing over time in the case of liquid H2O2, which is a refrigerated storage reagent, through the use of solid Na2O2.

[0067] As an example, the TMB strengthening pad (170) is such that when 150 ul (approximately 1 drop) of a sample diluted in an assay buffer of pH 8.5 is injected onto the sample pad (120), an acidic substance of pH 4.0 (e.g., a mixture of citric acid, etc.) is released in about 3 to 10 minutes, and the TMB strengthening reaction can proceed.

[0068]

[0069] 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.

[0070]

[0071] Manufacturing Example 1. Manufacturing of a lateral flow membrane sensor

[0072] As a membrane pad, Sartorius' Clear backed CN95 (25 mm) nitrocellulose was used and cut to a length of 30 cm.

[0073] Polyclonal anti-rabbit IgG was prepared at 1 mg / ml using PBS as a control line on the membrane pad, and monoclonal anti-mouse IgG was prepared at 1 mg / ml using PBS. These were then mixed and immobilized at 37°C.

[0074] An antibody (Anti-Human SARS-CoV-2 polyclonal antibody from Fapon) that specifically binds to COVID-19 was diluted to 1 mg / ml with PBS and prepared as a test line on the membrane pad. The antibody that specifically binds to COVID-19 (CoV) was immobilized and placed 5 mm apart from the control line. Each test line was coated with AppliCoat Plate Stabilizer from Applichem (AppliCoat Plate Stabilizer; Applichem #A7708) and dried at 37°C.

[0075] The sample pad was cut from Boreda's Grade 8964 (glass fiber), pretreated with Casein solution, dried at 37°C, and cut to a width and length of 4 x 18 mm. The absorbent pad was cut from Boreda's Grade 222 (glass fiber) to a width and length of 4 x 18 mm.

[0076]

[0077] MDI's PTR5 pad was prepared as a conjugate pad, and platinum nanoparticles (PtNP, 100 nm), which are chromogenic particles, were prepared by conjugating them with antibodies that specifically bind to COVID19 and blocking them with skim milk.

[0078] As a TMB reinforced pad (CA pad), Boreda's Grade 222 is prepared with a length shorter than the length of the sample pad, and citric acid (concentration 0.25 M), dextran sulfate (concentration 20 mg / ml), and Na2O2 (concentration 0.2 M) are mixed in a ratio of 8:1:1 (w:w:w) and dried at 37°C in a small-sized Grade 222.

[0079] As a TMB pad, a PTR5 pad from MDI was prepared. TMB (3,3',5,5'-Tetramethylbenzidine) was completely dissolved in DMSO at 15 mg / ml and then dried on the PTR5 pad at 37°C.

[0080]

[0081] A lateral flow membrane sensor is manufactured by positioning a membrane pad on a support made of PET material (polyethylene terephthalate, PET), positioning a TMB pad on one end of the membrane pad, positioning an absorption pad on the other end of the membrane pad, positioning a conjugate pad on one end of the TMB pad, positioning a sample pad on one end of the conjugate pad, and positioning a TMB reinforced pad (CA pad) on one end (end) of the sample pad.

[0082]

[0083] Comparative Example 1.

[0084] A lateral flow membrane sensor was manufactured by performing the same procedure as in Manufacturing Example 1 above, but excluding the TMB pad and the TMB reinforced pad, and positioning the conjugate pad on one end of the membrane pad.

[0085]

[0086] Experimental Example 1. Color reaction signal analysis

[0087] For each lateral flow membrane sensor manufactured in the above Manufacturing Example 1 and Comparative Example 1, 0.8 μl of capture antibody was immobilized as a circular spot, and then 3.8×10 3 TCID 50 100 μL of SARS-CoV-2 antigen was added dropwise. Afterwards, the intensity of the primary antigen-antibody reaction and the signal enhancement reaction of TMB were quantitatively observed over time, and the results are shown in Figure 4.

[0088] Referring to Fig. 4, in the TMB unreinforced structure (Comparative Example 1), the color development reaction slowly increases within about 15 minutes, whereas in the TMB reinforced structure (Manufacturing Example 1), the color development reaction occurs significantly between 3 and 10 minutes, and an intensity difference of about 2 to 2.5 times is observed.

[0089]

[0090] Experimental Example 2. Signal intensity comparison according to the location of the TMB pad and TMB reinforcement pad.

[0091] The lateral flow membrane sensor manufactured in the above Manufacturing Example 1 was included, and the TMB enhancement signal effect according to the presence and location of the TMB pad or TMB enhancement pad as a control was analyzed, and 3.8×10 3 TCID 50 100 μL of SARS-CoV-2 antigen was added dropwise to the sample, and the results of the analysis 20 minutes after sample injection (dropping) are shown in Figure 5.

[0092] Referring to FIG. 5, “⑤” is a lateral flow membrane sensor manufactured in the above Manufacturing Example 1, in which, when a sample is injected, the conjugate is first released and moves bypassing the TMB pad, and the primary reaction, which is an antigen-antibody reaction, initially occurs, and a portion of the sample is mixed with a dried CA mixture in the TMB enhanced pad (CA pad), and as the pH is lowered and the reaction progresses again with a time difference, the color-enhancing reaction of platinum nanoparticles (PtNPs) by TMB occurs secondarily.

[0093] "①" is a structure without a TMB pad, "②" is a structure in which a TMB pad is implemented in the form of a tape (TMP Tape) and is positioned on the test line and the control line, where the CA mixture is subsequently released from the TMB reinforced pad (CA pad) and reacts while the TMB is in direct contact with the PtNP, "③" is a structure in which a TMB reinforced pad (CA pad) is vertically stacked on the TMB pad, where the CA mixture is released in a vertical structure after the first reaction occurs, "④" is a structure in which the TMB pad is positioned between the conjugate pad and the sample pad, and "⑥" is a structure in which the CA mixture is directly dried on the sample pad, where the CA mixture is immediately released along with the sample injection, and the optimal pH change does not occur, so the second reaction does not occur.

[0094]

[0095] Experimental Example 3. Signal intensity comparison according to the combination of sample pad and absorbent pad.

[0096] In the case of Boreda's Grade 8964 (“8964”) used in the sample pad of the lateral flow membrane sensor manufactured in the above Manufacturing Example 1 and Boreda's Grade 222 (“AP22”) used in the absorbent pad (“8694-AP22”), as a control group (except for the types of sample pad and absorbent pad, the other conditions are the same), Boreda's Grade 222 (“AP22”) as the sample pad and Boreda's Grade 440 (“AP44”) as the absorbent pad (“AP22-AP44”), as a sample pad and Boreda's Grade 8964 (“8964”) as the sample pad and Boreda's Grade 440 (“AP44”) as the absorbent pad (“8964-AP44”), 3.8×10 were prepared on the sample pad. 3 TCID 50 100 μL of SARS-CoV-2 antigen was added dropwise to the sample, and the results of the analysis 20 minutes after sample injection (dropping) are shown in Figure 6.

[0097] Referring to FIG. 6, it can be confirmed that Boreda's Grade 8964 (“8964”) used in the sample pad of the lateral flow membrane sensor manufactured in Manufacturing Example 1 and Boreda's Grade 222 (“AP22”) used in the absorption pad (“8694-AP22”) exhibit excellent signal strength.

[0098]

[0099] Experimental Example 4. Signal intensity comparison according to TMB pad type

[0100] After preparing each membrane sensor manufactured using MDI's PTR5 pad (“PTR5”) used as the TMB pad of the lateral flow membrane sensor manufactured in the above Manufacturing Example 1, and Boreda's 6613, 6614, 8950, and 8951 as the TMB pad as the control group (the other conditions are the same except for the type of TMB pad), 3.8×10 2 3.8×10 4 TCID50 100 μL of SARS-CoV-2 antigen was added dropwise to the sample, and the results of the analysis 20 minutes after sample injection (dropping) are shown in Figure 7.

[0101] Referring to FIG. 7, it can be confirmed that excellent signal strength is exhibited when the PTR5 pad from MDI, which was used as the TMB pad of the lateral flow membrane sensor manufactured in Manufacturing Example 1, is used (“PTR5”).

[0102]

[0103] Manufacturing Example 2-1. Manufacturing of a lateral flow membrane sensor

[0104] The same procedure as in Manufacturing Example 1 was followed, but Citric acid (concentration 0.1 M) was used in the TMB reinforced pad (CA pad).

[0105]

[0106] Manufacturing Example 2-2. Manufacturing of a Lateral Flow Membrane Sensor

[0107] The same procedure as in Manufacturing Example 1 was performed, but Citric acid (concentration 0.125 M) was used in the TMB reinforced pad (CA pad).

[0108]

[0109] Manufacturing Example 2-3. Manufacturing of a Lateral Flow Membrane Sensor

[0110] The same procedure as in Manufacturing Example 1 was followed, but Citric acid (concentration 0.5 M) was used in the TMB reinforced pad (CA pad).

[0111]

[0112] Experimental Example 5. Signal intensity comparison according to citric acid concentration

[0113] For each of the lateral flow membrane sensors of Comparative Example 1, Manufacturing Example 1, Manufacturing Example 2-1 to Manufacturing Example 2-3, 3.8×10 3 TCID 50 / mL of SARS-CoV-2 antigen was added dropwise, and the results of the analysis 20 minutes after sample injection (dropping) are shown in Figure 8. In Figure 8, Comparative Example 1 is indicated as “w / o TMB Enh.” or “w / o Enh.”, Manufacturing Example 1 is indicated as “0.25 M,” Manufacturing Example 2-1 is indicated as “0.1 M,” Manufacturing Example 2-2 is indicated as “0.125 M,” and Manufacturing Example 2-3 is indicated as “0.5 M.”

[0114] Referring to Fig. 8, it can be confirmed that the signal intensity of the lateral flow membrane sensor of Manufacturing Example 1 with a citric acid concentration of 0.25 M is the best.

[0115]

[0116] Manufacturing Example 3-1. Manufacturing of a lateral flow membrane sensor

[0117] The same procedure as in Manufacturing Example 1 was followed, but malic acid (concentration 0.1 M) was used in the TMB reinforced pad (CA pad).

[0118]

[0119] Manufacturing Example 3-2. Manufacturing of a Lateral Flow Membrane Sensor

[0120] The same procedure as in Manufacturing Example 1 was followed, but maleic acid (concentration 0.1 M) was used in the TMB reinforced pad (CA pad).

[0121]

[0122] Manufacturing Example 3-2. Manufacturing of a Lateral Flow Membrane Sensor

[0123] The same procedure as in Manufacturing Example 1 was followed, but fumaric acid (concentration 0.1 M) (in DMSO) was used in the TMB reinforced pad (CA pad).

[0124]

[0125] Experimental Example 6. Signal intensity comparison according to the type of TMB reinforced pad acid

[0126] For each of the lateral flow membrane sensors of Comparative Example 1, Manufacturing Example 2-1, Manufacturing Example 3-1 to Manufacturing Example 3-3, 3.8 on the sample pad 3.8×10 4 TCID 50 / mL of SARS-CoV-2 antigen was added dropwise, and the results of the analysis 20 minutes after sample injection (dropping) are shown in Figure 9. In Figure 9, Comparative Example 1 is indicated as “w / o TMB Enh.” or “w / o Enh.”, Manufacturing Example 2-1 is indicated as “Citric acid”, Manufacturing Example 3-1 is indicated as “Malic acid”, Manufacturing Example 3-2 is indicated as “Maleic acid”, and Manufacturing Example 3-3 is indicated as “Fumaic acid” or “Fumaic acid in DMSO”.

[0127] Referring to Fig. 9, it can be confirmed that the signal intensity of the lateral flow membrane sensor of Manufacturing Example 2-1 using citric acid in the TMB reinforced pad is the best.

[0128]

[0129] Manufacturing Example 4-1. Manufacturing of a lateral flow membrane sensor

[0130] The same procedure as in Manufacturing Example 1 was performed, but Na2O2 (concentration 10 mM) was used.

[0131]

[0132] Manufacturing Example 4-2. Manufacturing of a Lateral Flow Membrane Sensor

[0133] The same procedure as in Manufacturing Example 1 was performed, but Na2O2 (concentration 50 mM) was used.

[0134]

[0135] Manufacturing Example 4-3. Manufacturing of a Lateral Flow Membrane Sensor

[0136] The same procedure as in Manufacturing Example 1 was performed, but Na2O2 (concentration 100 mM) was used.

[0137]

[0138] Comparative Example 2. Fabrication of a lateral flow membrane sensor

[0139] The same procedure as in Manufacturing Example 1 was followed, but H2O2 (concentration 10 mM) was used.

[0140]

[0141] Experimental Example 7. Signal intensity comparison according to Na2O2 concentration

[0142] For the lateral flow membrane sensors of Comparative Example 1, Comparative Example 2, Manufacturing Example 1, and Manufacturing Examples 4-1 to 4-3, 3.8×10 to 3.8×10 on the sample pad 4 TCID 50 / mL of SARS-CoV-2 antigen was added dropwise, and the results of analysis 20 minutes after sample injection (dropping) are shown in Fig. 10. In Fig. 10, Comparative Example 1 is indicated as “w / o Enh.”, Comparative Example 2 is indicated as “10 mM H2O2”, Manufacturing Example 1 is indicated as “20 mM Na2O2”, Manufacturing Example 4-1 is indicated as “10 mM Na2O2”, Manufacturing Example 4-2 is indicated as “50 mM Na2O2”, and Manufacturing Example 4-3 is indicated as “100 mM Na2O2”.

[0143] Referring to Fig. 10, it can be confirmed that the membrane sensors manufactured in Manufacturing Example 1 and Comparative Example 2 exhibit excellent signal strength.

[0144]

[0145] Experimental Example 8. Signal Response According to Temperature Conditions

[0146] For the lateral flow membrane sensor of the above Comparative Example 1 (w / o Enh.) and the above Manufacturing Example 1 (w / Enh.), 3.8 to 3.8×10 4 TCID 50 100 u㎕ of SARS-CoV-2 antigen was added dropwise at / mL, and the signal intensity under the measurement (sensor response) temperature conditions 20 minutes after sample injection (dropping) was analyzed and shown in Figure 11.

[0147] Referring to Fig. 11, it can be confirmed that excellent signal strength is observed under conditions of 28°C (Manufacturing Example 1), and a phenomenon of the membrane pad drying exists under conditions of 37°C (Manufacturing Example 5-2).

[0148]

[0149] Experimental Example 9. Signal Response According to Time-lapse Conditions

[0150] For the lateral flow membrane sensors of Comparative Example 1 (w / o Enh.) and Manufacturing Example 1 (w / Enh.), 3.8 to 3.8×10 were observed on the sample pad after the storage period of FIG. 12. 4 TCID 50 100 μL of SARS-CoV-2 antigen was added dropwise to the sample, and the signal intensity was analyzed 20 minutes after the addition, and the results are shown in Figure 12.

[0151] Referring to Figure 12, it can be seen that excellent signal strength is maintained even after long-term storage.

[0152]

[0153] Manufacturing Example 5-1. Manufacturing of a lateral flow membrane sensor

[0154] The same procedure as in Manufacturing Example 1 was followed, but an antibody that specifically binds to Influenza A (Flu A) was diluted to 1 mg / ml using PBS and prepared on the test line on the membrane pad, and then the antibody that specifically binds to Influenza A (Flu A) was immobilized.

[0155]

[0156] Manufacturing Example 5-2. Manufacturing of a Lateral Flow Membrane Sensor

[0157] The same procedure as in Manufacturing Example 1 was followed, but an antibody that specifically binds to Influenza B (Flu B) was diluted to 1 mg / ml using PBS and prepared on the test line on the membrane pad, and then the antibody that specifically binds to Influenza B (Flu B) was immobilized.

[0158]

[0159] Manufacturing Example 5-3. Manufacturing of a Lateral Flow Membrane Sensor

[0160] The same procedure as in Manufacturing Example 1 was followed, but an antibody that specifically binds to Adnovirus (AdV) was diluted to 1 mg / ml using PBS and prepared on the test line on the membrane pad, and then the antibody that specifically binds to Adnovirus (AdV) was immobilized.

[0161]

[0162] Manufacturing Example 5-4. Manufacturing of a Lateral Flow Membrane Sensor

[0163] The same procedure as in Manufacturing Example 1 was followed, but an antibody that specifically binds to RSV was diluted to 1 mg / ml using PBS and prepared on the test line on the membrane pad, and then an antibody that specifically binds to Adnovirus (AdV) was immobilized.

[0164]

[0165] Experimental Example 9. Analysis of color reaction signals according to antigen type

[0166] After preparing the lateral flow membrane sensors (w / TMB Enh.) manufactured in the above Manufacturing Examples 5-1 to 5-4 and the membrane sensors (w / o TMB Enh.) without the TMB reinforced pad in the above Manufacturing Examples 5-1 to 5-4 as a control group, 3.8×10 were placed on the sample pad for each membrane sensor. 1 3.8×10 5 TCID 50100 μL of SARS-CoV-2 antigen was added dropwise at / mL, and the signal intensity was analyzed 20 minutes after sample injection (dropping), and is shown in Figures 13a and 13b.

[0167] Referring to FIGS. 13a and 13b, all of the membrane sensors manufactured in Manufacturing Examples 5-1 to 5-4 had improved visual acuity, and were able to distinguish even very low concentrations that could not be detected with the naked eye when using quantitative equipment.

[0168]

[0169] Manufacturing Example 6. Manufacturing of a lateral flow membrane sensor

[0170] The same procedure as in Manufacturing Example 1 was followed, but an antibody specifically binding to RSV (Anti-Human RSV monoclonal antibody from Fapon) was diluted to 1 mg / ml using PBS and immobilized on a prepared membrane pad as a first test line, and an antibody specifically binding to COVID-19 (CoV) (Anti-Human SARS-CoV-2 polyclonal antibody from Fapon) was immobilized as a second test line, with the first and second test lines spaced at a distance of 3 mm.

[0171]

[0172] Experimental Example 10. Verification of Multiple Diagnostic Sensors

[0173] For the lateral flow membrane sensor manufactured in the above manufacturing example 6, 3.8 to 3.8×10 4 TCID 50 When 100 μL of SARS-CoV-2 antigen was dropped on the sample pad, 1.26 to 3.8 × 10 4 TCID 50 For the case where 100 μL of RSV-Type-B antigen was dropped at / mL, the signal intensity was analyzed 20 minutes after sample injection (dropping) and is shown in Figure 14.

[0174] Referring to Figure 14, it can be confirmed that a signal is expressed for each antigen.

[0175]

[0176] 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

sample pad; absorbent pad; A membrane pad disposed between the sample pad and the absorption pad; A conjugate pad disposed between the sample pad and the membrane pad; A TMB pad disposed between the membrane pad and the conjugate pad; and Including a TMB strengthening pad placed on the above sample pad, Lateral flow membrane sensor. In the first paragraph, The test line of the above membrane contains an antibody that specifically binds to any one of COVID19 (CoV), AdV (Adnovirus), Influenza A and Influenza B. Lateral flow membrane sensor. In the first paragraph, The above conjugate pad comprises platinum nanoparticles (Pt NPs). Lateral flow membrane sensor. In the first paragraph, The above TMB reinforcement pad is shorter than the above sample pad, Lateral flow membrane sensor. In paragraph 4, The above TMB reinforcement pad is placed on one side of the sample pad, Lateral flow membrane sensor. In the first paragraph, The above TMB reinforcing pad contains any one of citric acid, malic acid, maleic acid and fumaric acid as an acid substance. Lateral flow membrane sensor. In paragraph 6, The concentration of the above acid substance is 0.01 to 0.5 M, Lateral flow membrane sensor. In the first paragraph, The above TMB reinforcing pad contains Na2O2, Lateral flow membrane sensor. In the first paragraph, The concentration of the above Na2O2 is 10 to 100 mM, Lateral flow membrane sensor.

Images