Immunoassay method based on carbon nanomaterial and method for detecting target antigen by using immunoassay method

Abstract

The invention discloses a device and a method for detecting a target antigen in a sample. The device comprises a container; a carbon nanomaterial; and an antibody coupled to a marker wherein the antibody is immobilized on the surface of a carbon nanomaterial wherein the antibody has a binding site of a target antigen, and wherein when the target antigen binds to the binding site of the antibody, the antibody coupled to the marker is separated from the carbon nanomaterial, and the antibody labeled by the marker bound to the target antigen generates a signal.

Description

[0001] Citation of Related Applications

[0002] This application claims priority and benefits to U.S. Provisional Application No. 63 / 357,892, filed with the U.S. Patent and Trademark Office on July 1, 2022. This application also claims priority and benefits to U.S. Patent Application No. 18 / 124,986, filed with the U.S. Patent and Trademark Office on March 22, 2023. This application incorporates the entire contents of the above two applications by reference herein. Technical Field

[0003] The present disclosure relates to the technical field of immunoassay for detecting and quantitatively analyzing targets in biological and / or chemical samples, and in particular to a novel device and method for detecting target antigens using carbon nanomaterials with labeled antibodies fixed on their surfaces. Background Art

[0004] Immunoassays are widely used for early diagnosis and accurate prognosis of human diseases. In addition, viruses, bacteria, and toxins can also be detected and monitored using immunoassays. Currently, immunoassays can be divided into two different methods, namely sandwich immunoassays and competitive immunoassays.

[0005] Sandwich immunoassays use capture antibodies and detection antibodies to quantify macromolecular organisms (or antigens), such as viruses, cells, proteins, and peptides. To increase the sensitivity of sandwich immunoassays, detection antibodies are often coupled to specific materials, such as enzymes (like horseradish peroxidase (HRP), alkaline phosphatase (AP)), fluorescent dyes, radioactive isotopes, gold nanoparticles, single-stranded or double-stranded DNA, carbon dots, or quantum dots. In order to improve the sensitivity, accuracy, and reliability of sandwich immunoassays, multiple washing steps are required. As the concentration of antigen in the sample increases, the signal generated by the detection antibody coupled to the labeled material will increase accordingly. In summary, sandwich immunoassays can be operated through time-consuming multiple steps to quantify specific antigens in a sample.

[0006] Competitive immunoassays are performed using a single antibody and a constant amount of antigen coupled to a labeling material that is the same as the labeling material to which the detection antibody applied in the sandwich immunoassay is bound. Competitive immunoassays are used to quantify and monitor small molecules (or antigens) such as hormones and chemicals. In order to accurately and reliably quantify the antigen in the sample, multiple washing procedures are also required. The antigen in the sample and the labeled antigen compete for binding with the single antibody coated on the solid surface. Therefore, as the concentration of antigen in the sample increases, the signal generated by the labeled antigen decreases proportionally. In summary, since the signal measured in the absence of antigen is too high to detect low concentrations of antigen, it is difficult to quantify trace levels of antigen, and the sensitivity of competitive immunoassays is not as good as that of sandwich immunoassays.

[0007] The background description provided herein is for the purpose of generally presenting the context of the present disclosure. Unless otherwise indicated herein, the materials described in this section are not prior art to the claims in this application and are not admitted to be prior art or suggestion of prior art by inclusion in this section. Summary of the invention

[0008] The object of the present disclosure is to provide a device for detecting a target antigen in a sample, a method for detecting a target antigen, and a method for quantifying the target antigen based on a novel immunoassay method using a dual action of a carbon nanomaterial and an antibody coupled to a marker capable of capturing and detecting a specific antigen in a sample.

[0009] The novel immunoassay in the present disclosure is hereinafter referred to as "integrated immunoassay" only to distinguish it from conventional immunoassay methods, and therefore, the term "integrated" does not limit the scope of the invention disclosed herein. In the integrated immunoassay, antibodies coupled to markers can react with specific antigens quickly and generate signals without the need for time-consuming procedures such as multiple immunoreactions and washing.

[0010] According to an exemplary embodiment, the present disclosure provides a device for detecting a target antigen in a sample, comprising: a container; a carbon nanomaterial; and an antibody coupled to a marker, wherein the antibody is immobilized on the surface of the carbon nanomaterial, wherein the antibody has a binding site for the target antigen, and wherein, when the target antigen binds to the binding site of the antibody, the antibody coupled to the marker is separated from the carbon nanomaterial, and the marker-labeled antibody (marker <antibody> antigen) that binds to the antigen (marker antigen) generates a signal. The signal can be increased in proportion to the increase of the antigen in the sample.

[0011] The carbon nanomaterial may be one or more selected from graphene, graphene oxide, reduced graphene oxide, carbon nanotubes and carbon nanotube oxides.

[0012] The antibody may be immobilized on the surface of the carbon nanomaterial through non-covalent π-π stacking interactions, and the distance between the antibody and the carbon nanomaterial is 10 nm or less.

[0013] The sample may be a biological fluid selected from the group consisting of serum, plasma, whole blood, sweat, urine and cerebrospinal fluid.

[0014] The signal emitted by the label is selected from the group consisting of bioluminescence, chemiluminescence, fluorescence, colorimetry, electrochemistry, electrochemiluminescence, radiometry, and light visible to the naked eye.

[0015] The marker may be a bioluminescent marker selected from the group consisting of luciferase, luciferin and a luciferin derivative.

[0016] The label can be selected from acridinium ester chemiluminescent detection, chemiluminescent detection using horseradish peroxidase (HRP) labeled antibodies, chemiluminescent detection using alkaline phosphatase (ALP) labeled antibodies, chemiluminescent detection using phenylglyoxal derivatives, and chemiluminescent detection labels using ODI chemiluminescent detection operated with antibodies coupled to luminescent dyes.

[0017] The label can be a colorimetric detection label selected from the following detections: colorimetric detection performed with an antigen-binding antibody coupled to HRP, colorimetric detection performed with an antigen-binding antibody coupled to ALP, colorimetric detection performed with an antigen-binding antibody coupled to β-galactosidase, and colorimetric detection performed with an antigen-binding antibody coupled to glucose oxidase.

[0018] The label can be selected from the following electrochemiluminescent detection labels: ruthenium complex (Ru(bpy)3 2+ )-labeled antibodies, nanoparticles (e.g., gold, platinum, silver) or N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and specific substrates (e.g., tripropylamine, H2O2) and electrodes.

[0019] The label may be an electrochemical detection label selected from the group consisting of an antibody labeled with an enzyme (eg, HRP, ALP, glucose oxidase, β-galactosidase), a specific substrate, and an electrode.

[0020] The label can be selected from the following fluorescent detection labels: (1) enzyme (e.g., HRP, ALP) labeled antibodies and specific substrates (e.g., Amplex Red, H2O2, fluorescein diphosphate (FDP), 4-methylumbelliferyl phosphate) and (2) fluorescent dye labeled antibodies.

[0021] The label can be selected from the following naked eye detectable labels: enzymes (eg, HRP, ALP) and specific substrates capable of color development (eg, Amplex Red, Zonyl FSN-100 functionalized gold nanoparticles).

[0022] The label may be a radioactive detection label selected from the following: isotopes (e.g., 125 I) Labeled antibodies.

[0023] According to another exemplary embodiment, the present disclosure provides a method for manufacturing the above-mentioned device, comprising: fixing the carbon nanomaterial on the inner surface of the container; providing the antibody coupled to the marker; and contacting the antibody coupled to the marker with the surface of the carbon nanomaterial.

[0024] The carbon nanomaterial may be magnetic graphene, graphene oxide and reduced graphene oxide, single-walled carbon nanotubes and multi-walled carbon nanotubes.

[0025] The carbon nanomaterial may be in the form of a thin film composed of graphene oxide, and the container is a polystyrene container.

[0026] According to another exemplary embodiment, the present disclosure provides a method for detecting a target antigen, comprising: introducing a sample solution into the above-mentioned device; and detecting a signal generated by an antibody labeled with the marker (marker <antibody> antigen) that binds to the target antigen to detect the target antigen in the sample solution.

[0027] The carbon nanomaterial may be one or more selected from graphene, graphene oxide, reduced graphene oxide, single-walled carbon nanotubes and multi-walled carbon nanotubes.

[0028] The carbon nanomaterial has a film form composed of graphene oxide, and the container is a polystyrene container.

[0029] The antibody may be immobilized on the surface of the carbon nanomaterial through non-covalent π-π stacking interactions, and the distance between the antibody and the carbon nanomaterial is 10 nm or less.

[0030] The sample solution may be a biological solution selected from serum, plasma, whole blood, sweat, urine and cerebrospinal fluid.

[0031] The signal generated by the marker-labeled antibody bound to the target antigen is selected from the group consisting of bioluminescence, chemiluminescence, fluorescence, colorimetry, electrochemistry, electrochemiluminescence, radiometry, and visible light.

[0032] The method may not use an artificially produced antigen coupled to the marker.

[0033] The method may not use a detection antibody conjugated to a label.

[0034] The method may include two or more types of antibodies.

[0035] The method may not include a washing procedure to remove waste after introducing the sample solution into the device.

[0036] The signal may be emitted within 30 minutes after the sample solution containing the target antigen is introduced into the device.

[0037] According to another exemplary embodiment, the present disclosure provides a method for quantifying a target antigen, comprising: introducing a sample solution into the above-mentioned device; and measuring the intensity of the signal to quantify the target antigen in the sample solution.

[0038] The carbon nanomaterial may be one or more selected from graphene, graphene oxide, reduced graphene oxide, single-walled carbon nanotubes and multi-walled carbon nanotubes.

[0039] The antibody may be immobilized on the surface of the carbon nanomaterial through non-covalent π-π stacking interactions, and the distance between the antibody and the carbon nanomaterial is 10 nm or less.

[0040] The sample solution may be a biological solution selected from serum, plasma, whole blood, sweat, urine and cerebrospinal fluid.

[0041] The signal emitted by the label is selected from the group consisting of bioluminescence, chemiluminescence, fluorescence, colorimetry, electrochemistry, electrochemiluminescence, radiometry and light visible to the naked eye.

[0042] The method may not use an artificially produced antigen coupled to the marker.

[0043] The method may not use a detection antibody coupled to the label.

[0044] The method may comprise two or more types of antibodies.

[0045] The method may not include a washing procedure to remove waste after introducing the sample solution into the device.When the concentration of the target antigen in the sample solution increases, the intensity of the signal may increase proportionally.

[0046] The signal may be emitted within 30 minutes after the sample solution is introduced into the device. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The accompanying drawings illustrate preferred embodiments of the present disclosure and, together with the foregoing disclosure, are used to provide a further understanding of the technical features of the present disclosure. Therefore, the present disclosure is not interpreted as being limited to the accompanying drawings.

[0048] According to one embodiment of the present disclosure, Figure 1 (A) shows the structure of the antibody, Figure 1 (B) shows the interaction between the antibody and carbon nanomaterial in the presence and absence of an antigen that strongly binds to the antibody.

[0049] Figure 2 A schematic diagram of the integrated immunoassay with chemiluminescent detection according to another embodiment of the present disclosure is shown.

[0050] Figure 3 shows a conventional sandwich immunoassay with chemiluminescent detection.

[0051] FIG. 4 shows a conventional competitive immunoassay with chemiluminescent detection.

[0052] Figure 5 A schematic diagram of the integrated immunoassay with ODI chemiluminescent detection for quantifying T4 in human serum according to another embodiment of the present disclosure is shown.

[0053] Figure 6 A schematic diagram of the integrated immunoassay with acridinium ester chemiluminescent detection for quantifying CA-19 in human serum according to another embodiment of the present disclosure is shown.

[0054] Figure 7 A schematic diagram showing bioluminescence detection using luciferase-labeled antibodies in the integrated immunoassay according to another embodiment of the present disclosure is shown.

[0055] Figure 8 Another embodiment according to the present disclosure is shown, which uses (a) 6-amino-D-fluorescein or (b) 6-aminoseleno-D-fluorescein labeled antibody-bound antigen for bioluminescent detection.

[0056] Fig. 9 An acridinium NHS ester derivative that can be used for the integrated immunoassay according to another embodiment of the present disclosure is shown.

[0057] Fig.10 The invention shows the use of (a) acridine C2, NHS ester or (b) Lumiwo in the integrated immunoassay according to another embodiment of the present disclosure. TM Mechanism of acridinium ester chemiluminescent reaction of acridinium NHS ester labeled antibodies.

[0058] Fig.11 The integrated immunoassay using acridinium ester linked to (a) fluorescein, (b) rhodamine B, and (c) BDP according to another embodiment of the present disclosure is shown.

[0059] Fig.12 The integrated immunoassay using luminol chemiluminescent detection of an antibody conjugated to HRP according to another embodiment of the present disclosure is shown.

[0060] Fig.13 The integrated immunoassay assay using luminol chemiluminescent detection of antibodies conjugated to ABEI according to another embodiment of the present disclosure is shown.

[0061] Fig.14The integrated immunoassay for chemiluminescent detection of ODI using an antibody coupled to HRP in the presence of Amplex Red according to another embodiment of the present disclosure is shown. 1: Amplex Red, 2: resorufin in the ground state, 3: resorufin in the excited state, 4: ODI, 5: high-energy intermediate formed by the reaction of ODI and H2O2.

[0062] Fig.15 The integrated immunoassay with chemiluminescent detection based on the reaction operation of 3-(2'-spiroadamantyl)-4-methoxy-4-(3"-phosphoryloxy)-phenyl-1,2-dioxetane (adamantyl 1,2-dioxetane aryl phosphate, AMPPD) (which is one of the adamantyl 1,2-dioxetane phosphate derivatives) and ALP according to another embodiment of the present disclosure is shown.

[0063] Fig.16 The integrated immunoassay with ODI chemiluminescent detection using ALP enzyme reaction in the presence of FDP according to another embodiment of the present disclosure is shown. 1: FDP, 2: fluorescein in the ground state, 3: fluorescein in the excited state, 4: ODI, X: high-energy intermediate formed by the reaction of ODI and H2O2.

[0064] Fig.17 The integrated immunoassay with ODI chemiluminescent detection using ALP enzyme reaction in the presence of 4-MUP according to another embodiment of the present disclosure is shown. 1: 4-MUP, 2: 4-MU in the ground state, 3: 4-MU in the excited state, 4: ODI, X: high-energy intermediate formed by the reaction of ODI and H2O2.

[0065] Fig.18 The target antigen bound to single-stranded DNA (20-100 bp) and an antibody coupled to a luminescent dye according to another embodiment of the present disclosure is shown: 1: Pacific Blue, 2: 6-carboxyfluorescein (6-FAM), 3: fluorescein.

[0066] Fig.19 The integrated immunoassay using chemiluminescent detection of phenylglyoxal derivatives according to another embodiment of the present disclosure is shown. 1: cytosine, 2: adenine, 3: guanine, 4: thymine, 5. Pacific blue in the ground state, 6: high energy intermediate formed by the reaction of guanine and TMPG (1-10 mM) in the presence of TPA (15-30 mM) and DMF, 7: Pacific blue in the excited state.

[0067] Fig. 20The multiplex integrated immunoassay using chemiluminescent detection of phenylglyoxal derivatives according to another embodiment of the present disclosure is shown, which can simultaneously quantify three different target antigens in a sample: 1: Pacific Blue, 2: 6-FAM, 3: Rhodamine 101 (or ROX).

[0068] Fig.21 The integrated immunoassay with ODI chemiluminescent detection is shown according to another embodiment of the present disclosure operating with an antibody coupled to Rhodamine 101 via (1) a linker and (2) single-stranded DNA.

[0069] Fig. 22 The multiplex integrated immunoassay with ODI chemiluminescent detection according to another embodiment of the present disclosure is shown, which can simultaneously quantify three different target antigens in a sample: 1: Pacific Blue, 2: 6-FAM, 3: Rhodamine 101 (or ROX).

[0070] Fig.23 The integrated immunoassay for colorimetric detection using an HRP-labeled antibody capable of capturing a target antigen and TMB (3,3',5,5'-tetramethylbenzidine) as a substrate is shown according to another embodiment of the present disclosure.

[0071] Fig.24 The integrated immunoassay for colorimetric detection using an HRP-labeled antibody capable of capturing a target antigen and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) as a substrate is shown according to another embodiment of the present disclosure.

[0072] Fig.25 The integrated immunoassay for colorimetric detection using an HRP-labeled antibody capable of capturing a target antigen and OPD (o-phenylenediamine dihydrochloride) as a substrate according to another embodiment of the present disclosure is shown.

[0073] Fig.26 The integrated immunoassay using an HRP-labeled antibody capable of capturing a target antigen and Amplex Red as a substrate for colorimetric detection according to another embodiment of the present disclosure is shown.

[0074] Fig. 27 The integrated immunoassay with colorimetric detection using an ALP-labeled antibody capable of capturing a target antigen and a pNPP (disodium p-nitrophenyl phosphate) substrate according to another embodiment of the present disclosure is shown.

[0075] Fig.28The integrated immunoassay using a β-galactosidase-labeled antibody capable of capturing a target antigen and colorimetric detection of ONPG (o-nitrophenyl-β-D-galactopyranoside) as a substrate according to another embodiment of the present disclosure is shown.

[0076] Fig.29 The integrated immunoassay for colorimetric detection using a glucose oxidase-labeled antibody capable of capturing a target antigen and TMB as a substrate according to another embodiment of the present disclosure is shown.

[0077] Fig.30 The integrated immunoassay according to another embodiment of the present disclosure uses an ALP-labeled antibody capable of capturing a target antigen and electrochemical detection of 3-indoxyl phosphate disodium salt as a substrate. 1: 3-indoxyl phosphate disodium salt, 2: indigo.

[0078] Fig.31 The integrated immunoassay using an HRP-labeled antibody capable of capturing a target antigen, H2O2, and electrochemical detection of acetaminophen as a substrate according to another embodiment of the present disclosure is shown. 1: acetaminophen, 2: N-acetyl-4-quinoneimine.

[0079] Fig.32 The integrated immunoassay using electrochemiluminescent detection according to another embodiment of the present disclosure is shown, and the electrochemiluminescent detection operates with an antibody labeled with a ruthenium complex that binds to a target antigen.

[0080] Fig.33 The integrated immunoassay using electrochemiluminescent detection according to another embodiment of the present disclosure is shown, and the electrochemiluminescent detection operates with antibodies labeled with gold nanoparticles (Au) that bind to target antigens.

[0081] Fig.34 The integrated immunoassay using electrochemiluminescent detection according to another embodiment of the present disclosure is shown, and the electrochemiluminescent detection operates with an ABEI-labeled antibody that binds to a target antigen.

[0082] Fig.35 The integrated immunoassay using enzyme-labeled antibodies and fluorescence detection operated by substrates according to another embodiment of the present disclosure is shown.

[0083] Fig.36 The integrated immunoassay using fluorescence detection performed using antibodies labeled with fluorescent dyes according to another embodiment of the present disclosure is shown.

[0084] Fig.37 The integrated immunoassay capable of sensing positive and negative samples with the naked eye according to another embodiment of the present disclosure is shown.

[0085] Fig.38 The integrated immunoassay using a radiometer capable of sensing radioactivity according to another embodiment of the present disclosure is shown.

[0086] Fig.39 Shown are (a) an image of pure graphene oxide observed using a transmission electron microscope (TEM), (b and c) images of magnetic graphene oxide obtained from TEM, and (d) separation of magnetic graphene oxide using a magnetic separator according to another embodiment of the present disclosure.

[0087] Fig.40 The separation of antibody-conjugated acridinium ester temporarily immobilized on magnetic carbon nanomaterials according to another embodiment of the present disclosure is shown. From left to right: graphene oxide, reduced graphene oxide, graphene, 10-20 nm multi-walled carbon nanotubes, 20-30 nm multi-walled carbon nanotubes, and 30-50 nm multi-walled carbon nanotubes.

[0088] Fig.41 CL / CLO determined by the integrated immunoassay operated with 6 different magnetic carbon nanomaterials according to another embodiment of the present disclosure is shown.

[0089] Fig.42 Shown are (a) a graphene oxide film fixed on the surface of a polystyrene pore, (b) reduced graphene oxide and a graphene film on the surface of a polystyrene pore, and (c) three different sizes of wet and dry multi-walled carbon nanotubes on a polystyrene pore according to another embodiment of the present disclosure.

[0090] Fig.43 A graphene oxide film is formed on the surface of polystyrene according to another embodiment of the present disclosure. From left to right: Tris-HCl (pH 7), Tris-HCl (pH 7.5), Tris-HCl (pH 8), Tris-HCl (pH 8.5), Tris-HCl (pH 9), TBS (pH 7.4), PBS (7.4), and sodium phosphate (pH 7).

[0091] Fig.44 The CL / CLO in the integrated immunoassay performed using graphene oxide membranes formed in various buffer solutions for quantifying D-dimer in human serum according to another embodiment of the present disclosure is shown.

[0092] Fig.45 The CL / CLO in the integrated immunoassay for quantifying D-dimer in human serum operated with graphene oxide membranes formed at various pH in sodium phosphate buffer according to another embodiment of the present disclosure is shown.

[0093] Fig.46 The CL / CLO in the integrated immunoassay for quantifying D-dimer in human serum operated with an antibody molecule coupled to an acridinium ester in various buffers according to another embodiment of the present disclosure is shown.

[0094] Fig.47 The CL / CLO measured by the integrated immunoassay using multiple detection methods such as bioluminescence, chemiluminescence, and electrochemiluminescence according to another embodiment of the present disclosure is shown.

[0095] Fig.48 Shown are (a) the correlation between acridine and luminol chemiluminescence used as the detection method of the integrated immunoassay, (b) the correlation between acridine and AMPPD chemiluminescence used as the detection method of the integrated immunoassay, and (c) the correlation between luminol and AMPPD chemiluminescence used as the detection method of the integrated immunoassay according to another embodiment of the present disclosure. DETAILED DESCRIPTION

[0096] Although the present disclosure allows for various modifications and alternative embodiments, specific embodiments thereof will be described and shown by way of example in the accompanying drawings. However, this does not mean that the present disclosure is limited to a specific disclosure form, but should be understood to include all modifications, equivalents and substitutes within the concept and technical scope of the present disclosure.

[0097] In the present application, it should be understood that terms such as “including” or “having” are intended to indicate the presence of the features, quantities, steps, operations, components, parts, or combinations thereof described in the specification, and they do not preclude the possibility of the presence or addition of one or more other features or quantities, steps, operations, components, parts, or combinations thereof.

[0098] Hereinafter, the present disclosure will be described in detail.

[0099] All types of antibodies can bind weakly but stably to carbon nanomaterials via non-covalent π-π stacking interactions in biological solutions such as serum, plasma, whole blood, and cerebrospinal fluid, as well as various samples related to the environment, public health, and food safety. Figure 1As shown in (a), in antibodies, there are multiple amine groups located on the antigen binding site. These amine groups of antibodies can interact non-covalently with carbon nanomaterials. (See S. Shahriari, M. Sastry, S. Panjikar and RK Singh Raman published in Nanotechnology, Science and Applications, Vol. 14, 2021, pp. 197-220. See also JG Wuo, L.-H. Tran, S.-H. Jang, C. Lee and TJ Kang published in Journal of the Korean Chemical Society, Vol. 36, 2015, pp. 2959-2961.).

[0100] Therefore, due to non-covalent π-π stacking interactions, the antigen binding site of the antibody can bind to the carbon nanomaterials, such as Figure 1 However, in the presence of antigen, the π-π stacking interaction between the specific antibody and the carbon nanomaterial is rapidly decomposed to strongly and permanently bind to the antigen in the solution, as shown in FIG. Figure 1 (b). When the antibody is immobilized on the surface of the carbon nanomaterial through π-π stacking interaction, the distance between the antibody and the carbon nanomaterial is shorter than 10 nm. Therefore, most of the signals generated by the antibody labeled with the marker (i.e., biological or chemical material) on the carbon nanomaterial are lost because the carbon nanomaterial absorbs the signal. For example, due to the rapid chemiluminescence (or fluorescence) resonance energy transfer from the biological or chemical material to the carbon nanomaterial, the intensity of the chemiluminescence (or fluorescence) emitted under such conditions is very weak. However, the chemiluminescence (or fluorescence) generated by the antibody labeled with the biological or chemical material, after being separated from the carbon nanomaterial through the binding interaction between the antibody and the antigen, is stronger in intensity and is not subject to any interference from the carbon nanomaterial. This is because the distance between the separated antibody and the carbon nanomaterial is greater than 10 nm, which is the maximum distance of the chemiluminescence (or fluorescence) resonance energy transfer between the biological or chemical material and the carbon nanomaterial. Therefore, the signal intensity depends on the concentration of the antigen in the sample. In other words, the signal intensity increases proportionally with the increase of the antigen in the sample. Based on Figure 1 Based on the phenomenon shown in , the inventors of the present disclosure invented a novel immunoassay as described in the present disclosure. In order to distinguish this novel immunoassay from conventional immunoassay technology, the novel immunoassay according to the present disclosure will be referred to as "integrated immunoassay" in this specification.

[0101] Figure 2The integrated immunoassay shown in FIG. 3 can rapidly quantify antigens in a sample without the time-consuming procedures (eg, multiple incubations and washes) required by conventional methods such as competitive and sandwich immunoassays shown in FIG. 3 and FIG. 4 . Figure 2 -4 is an example showing the integrated immunoassay compared with a conventional immunoassay using chemiluminescent detection (e.g., alkaline phosphatase, horseradish peroxidase (HRP) or acridinium ester chemiluminescent detection). In addition, the integrated immunoassay can be used with various detection methods (e.g., bioluminescence, chemiluminescence, colorimetry, electrochemistry, electrochemiluminescence, fluorescence, and radiometry) by simply replacing the antibody labeled with the biological or chemical material. In summary, the integrated immunoassay using specific antibodies coupled to biological or chemical materials performs faster and better than conventional immunoassays in terms of analysis time and sensitivity, and the biological or chemical materials are used to generate signals such as bioluminescence, chemiluminescence, colorimetry, electrochemistry, electrochemiluminescence, fluorescence, and radiometry. This is because the integrated immunoassay is performed without adding (1) artificially manufactured antigens coupled to biological or chemical materials for competitive immunoassays and (2) detection antibodies coupled to biological or chemical materials (i.e., markers) for sandwich immunoassays. In addition, the signal measured from the integrated immunoassay always increases with the increase of the concentration of the specific antigen in the sample, because the concentration of the antibody separated from the carbon nanomaterial surface is proportional to the amount of antigen in the sample. For example, the 1,1'-oxalyldiimidazole (ODI) chemiluminescence emitted from the rapid integrated immunoassay using HRP-labeled antibodies weakly immobilized on the surface of graphene oxide membranes increases with the increase of thyroxine (T4) in the sample, as shown in FIG. Figure 5 As shown, the 1,1'-oxalyldiimidazole (ODI) chemiluminescence generated from a time-consuming competitive immunoassay with luminol chemiluminescence detection, which is used as a conventional immunoassay for analyzing small molecules such as T4, decreases exponentially with increasing T4 concentration. The integrated immunoassay capable of quantifying T4 within 25 minutes is more sensitive than the commercially available competitive immunoassay (T4 Acculite CLIA, Monobind, Inc.) that operates with a 50-minute incubation and multiple washes. In addition, using the integrated immunoassay with acridinium ester chemiluminescence detection, the analysis time for quantifying CA 19-9 (which is a tumor marker) is as short as 30 minutes. On the other hand, the commercially available conventional sandwich immunoassay with luminol chemiluminescence detection (CA 19-9 Acculite CLIA kit, Monobind, Inc) requires multiple incubations and several time-consuming washes that take a total of 80 minutes to analyze CA 19-9. In terms of quantifying CA 19-9 in human serum, Figure 6 The sensitivity of the integrated immunoassay with a good linear calibration curve shown in is superior to that of the time-consuming sandwich immunoassay.

[0102] The integrated immunoassay can be operated using all detection methods applied to conventional competitive and sandwich immunoassays. Therefore, antigens bound to antibodies labeled with biological or chemical materials obtained from the integrated immunoassay can be quantified using various detection methods, such as bioluminescence, chemiluminescence, colorimetry, electrochemistry, electrochemiluminescence, naked eye, fluorescence, and radiometric determination. The details are as follows.

[0103] A. Detection Methods Used in the Integrated Immunoassay

[0104] The integrated immunoassay is based on one of several antibody labeling methods (e.g., NHS (succinimidyl) ester method, carbodiimide method, and periodate method), wherein the specific antibody used in the integrated immunoassay is coupled to a label capable of generating a signal. These methods for labeling labels into antibodies are well known in the art, for example, are described in the following documents, which are incorporated herein by reference.

[0105] *E. Ishikawa, Immunoassay, EP Diamandis and TK Christopoulos, eds., Academic Press, San Diego, 1996, DOI: https: / / doi.org / 10.1016 / B978-012214730-2 / 50009-1 ,pp.191-204.

[0106] *H. Jiang, GDD'Agostino, PACole., and DR Dempsey, in Methods in Enzymology, DM Chenoweth, ed., Academic Press, 2020, 639, 333-353.

[0107] 1. Integrated immunoassay and bioluminescence detection

[0108] like Figure 7 and 8 As shown, the antibody used in the integrated immunoassay can be coupled to luciferase or a luciferin derivative such as 6-amino-D-luciferin, and 6-aminoseleno-D-luciferin can emit bioluminescence in the presence of an antigen that can strongly bind to the antibody. The intensity of the bioluminescence emitted by the integrated immunoassay increases with the increase of the antigen in the sample. For example, the antigen bound to the antibody coupled to luciferase increases with the addition of a luciferin derivative, Mg 2+The invention discloses a method for detecting the bioluminescence of an integrated immunoassay in the presence of a 50 mM Tris-HCl (50 mM, pH 8.5) solution containing adenosine triphosphate (ATP). The color of the bioluminescence in the integrated immunoassay is determined by the properties of luciferin derivatives. The bioluminescence in the presence of potassium D-luciferin, 6-amino-D-luciferin or 6-aminoseleno-D-luciferin is yellow, yellow-orange or red. In addition, the antigen bound to the antibody coupled to 6-amino-D-luciferin or 6-aminoseleno-D-luciferin is brightly emitted when a certain concentration of luciferase, Mg 2+ ... 2+ The integrated immunoassay can quantify the target antigen with good accuracy, precision and reliability by adjusting the concentration of the reagent used to generate bioluminescence in the integrated immunoassay. For example, using 0.5 mM 6-amino-D-luciferin, 10 mM Mg 2+ and 5 mM ATP to quantify trace levels of D-dimer in human serum. Immediately after adding the three reagents, the integrated immunoassay measures the bioluminescence in the integrated immunoassay for 5 seconds.

[0109] 2. Integrated immunoassay and chemiluminescence detection

[0110] 2.1 Integrated immunoassay with acridinium ester chemiluminescence detection

[0111] There are several types of acridinium ester labeled antibodies that can be used in the integrated immunoassay. For example, Fig. 9 The two different acridinium ester derivatives shown can be labeled in antibodies, e.g. Fig.10 As shown. Both acridinium ester derivatives dissolved in DMSO can bind to the primary amines of the antibody within 30 minutes at room temperature. Fig.10 As shown in (A), a high-energy intermediate (or activator) formed by the sequential addition of (1) H2O2 (5-40 mM) in HNO3 (25-100 mM) and (2) NaOH (0.1-0.5 N) containing trace concentrations of Triton X-100 is separated from the antibody. The high-energy intermediate then emits bright blue chemiluminescence. In addition, after the sequential addition of the two reagents, a high-energy intermediate that binds to the antibody is also formed, as shown in FIG. Fig.10 (b) is shown. The high-energy intermediate also emits blue chemiluminescence. Fig.10The acridinium ester chemiluminescence in the integrated immunoassay operated by the reaction mechanism shown in is proportionally enhanced with the increase of D-dimer in human serum. Based on the chemiluminescence resonance energy transfer (CRET) principle of the high-energy intermediate formed from the chemical reaction to the luminescent dye, the integrated immunoassay using acridinium ester to be connected with the luminescent dye (e.g., fluorescein, rhodamine B, BDP), its emission wavelength is longer than the emission wavelength when using acridinium C2 NHS ester. Therefore, in the integrated immunoassay, the color of the chemiluminescence emitted from the acridinium ester connected with the luminescent dye is determined by the emission wavelength of the luminescent dye. In short, the integrated immunoassay can use the antibody combined with 2-4 kinds of luminescent dyes to analyze the multiple target antigens present in the sample at the same time, and these luminescent dyes can emit chemiluminescence of different colors, such as blue, green, yellow-orange and red. In other words, the integrated immunoassay using the luminescent dye connected with acridinium ester can be used as a new multiple determination method for quantifying several target antigens in the sample at the same time.

[0112] 2.2 Integrated immunoassay with horseradish peroxidase chemiluminescence detection

[0113] 2.2.1. Luminol chemiluminescence detection integrated immunoassay

[0114] Luminol chemiluminescence can be used as a detection method for the integrated immunoassay. Fig.12 As shown, when an antibody coupled to horseradish peroxidase (HRP) is placed in a Tris-HCl (10-100 mM, pH 8-9) solution containing an enhancer (such as 4-iodophenol, 4-(16-imidazol-1-yl)phenol, 4-bromophenol, 4-phenylphenol, 3-(10'-phenothiazinyl)propane-1-sulfonate and 4-morpholinopyridine), and hydrogen peroxide (1-5 mM) and luminol (or isoluminol, 0.1-2 mM) are added, blue chemiluminescence is emitted. In addition, the integrated immunoassay can also be performed with an antibody coupled to N-(4-aminobutyl)-N-ethylisoluminol (ABEI), which can emit bright luminol chemiluminescence when HRP (0.5-10 U / ml) and H2O2 (1-10 mM) are added to a Tris-HCl (10-100 mM, pH 8-9) solution containing the above-mentioned enhancers (such as 4-iodophenol, 4-(16-imidazol-1-yl)phenol, 4-bromophenol, 4-phenylphenol, 3-(10'-phenothiazinyl)propane-1-sulfonate and 4-morpholinopyridine). Fig.13 For example, using Fig.12 and Fig.13 The integrated immunoassay with two different luminol chemiluminescent detection methods shown in Figure 1 can quantify trace levels of D-dimer in human serum.

[0115] 2.2.2.1,1′-oxalyldiimidazole chemiluminescence detection HRP enzyme reaction integrated immunoassay

[0116] like Fig.14 As shown, the HRP-bound antigen coupled to the antibody can be quantified using the integrated immunoassay with 1,1'-oxalyldiimidazole (ODI) chemiluminescent detection. The HRP-labeled antibody reacts with Amplex Red (a non-luminescent dye, 5-50 μM) in Tris-HCl (5-100 mM, pH 7-8.5) or PBS (10 mM, pH 7.4) in the presence of H2O2 (0.5-2 mM) and an enhancer (e.g., 4-iodophenol, 4-(imidazol-1-yl)phenol, 4-bromophenol, 4-phenylphenol) to produce resorufin as a luminescent dye. The incubation time range (1-10 minutes) of the reaction depends on the experimental conditions of the integrated immunoassay. For example, the incubation time for quantification of the COVID-19 receptor binding domain (RBD) in nasal specimens is 5 minutes, while the incubation time for quantification of D-dimer in human serum is 3 minutes. Fig.14 The resorufin generated by the HRP enzyme reaction shown emits bright red chemiluminescence in a mixed solution of isopropanol and ODI (generated by the reaction of 2,4,6-trichlorophenyl oxalate (TCPO, 10-100M) with an imidazole derivative (20-400M), such as imidazole, 2-methylimidazole, 2,4-dimethylimidazole, 4-methylimidazole and 2-ethylimidazole in ethyl acetate) with the continuous addition of H2O2 (20-100mM). The brightness of the chemiluminescence emitted in the integrated immunoassay with ODI-CL detection increases with the increase of the target antigen concentration in the sample.

[0117] 2.3. Integrated immunoassay using chemiluminescent detection of alkaline phosphatase (ALP)-labeled antibodies

[0118] 2.3.1. Integrated immunoassay with alkaline phosphatase-labeled antibody-induced chemiluminescence of substrate

[0119] Adamantyl 1,2-dioxetane phosphate derivatives, such as adamantyl 1,2-dioxetane aryl phosphate (AMPPD) and adamantyl 1,2-dioxetane chlorophenyl phosphate (CSPD), emit glow chemiluminescence after reacting with alkaline phosphatase (ALP) under alkaline conditions (pH 9-11). Fig.15 As shown, AMPPD, 1 reacts with the ALP-labeled antibody-bound antigen formed in the integrated immunoassay to produce intermediate 2. Then, intermediate 2 is rapidly separated into high-energy intermediate 3 and stable product 4 in an excited state. Finally, high-energy intermediate 3 emits blue chemiluminescence. For example, using a method based on Fig.15 The integrated immunoassay with chemiluminescent detection operated by the mechanism shown in is used to quantify trace levels of D-dimer in human serum. The brightness of the chemiluminescence emitted by the integrated immunoassay increases with the increase of D-dimer in human serum. The mixture of ALP-bound antigen coupled to the antibody and the CSPD derivative is incubated for 5 minutes, and then the intensity of the chemiluminescence emitted in the detection unit is measured for 5 seconds using a reliable method.

[0120] 2.3.2. ODI Chemiluminescence Integrated Immunoassay Operated by ALP Enzyme Reaction

[0121] Fluorescein diphosphate (FDP) and 4-methylumbelliferylphosphate (4-MUP) are non-luminescent compounds. However, in the presence of alkaline phosphatase (ALP), FDP and 4-MUP are hydrolyzed to produce fluorescein and 4-methylumbelliferon (4-MU), which are strong luminescent dyes. The yield of fluorescein and 4-MU converted from FDP and 4-MUP depends on the activity of ALP added for the enzyme reaction. Therefore, based on Fig.16 and 17 The mechanism shown in the integrated immunoassay with ODI chemiluminescent detection can be applied to quantify the target antigen in the sample. The color of ODI chemiluminescence depends on the properties of the luminescent dye from the ALP enzyme reaction in the presence of a non-luminescent compound. For example, ALP (alkaline phosphatase) coupled to an antibody is used to quantify the trace level of D-dimer in human serum. The enzyme can specifically capture D-dimer and fibrin degradation products (FDP) in the sample and detect it using ODI chemiluminescent detection. After 15 minutes of binding reaction between D-dimer and the antibody coupled to ALP in the integrated immunoassay kit, FDP (0.1-0.5mM) in Tris-HCl (10mM, pH 8.5) is placed in the kit and reacted for 5 minutes to produce fluorescein. After the final incubation, the chemiluminescence of ODI generated by the reaction of 2,4,6-trichlorophenyl oxalate (TCPO, 10-100 μM) with imidazole derivatives (20–400 μM) (such as imidazole, 2-methylimidazole, 2,4-dimethylimidazole, 4-methylimidazole, and 2-ethylimidazole) in ethyl acetate was measured for 2 seconds by sequential addition of H2O2 (20-100 mM) to isopropanol and ODI. The relative chemiluminescence intensity increases with increasing concentrations of D-dimer in human serum.

[0122] 2.3 Integrated immunoassay for chemiluminescence detection of phenylglyoxal derivatives

[0123] Phenylglyoxal derivatives, such as 3-methoxyphenylglyoxal (3-MPG) and 3,4,5-trimethoxyphenylglyoxal (TMPG), can react with guanine in single-stranded DNA (or RNA) to produce high-energy intermediates, which can emit weak chemiluminescence themselves and can also transfer energy to luminescent dyes based on the principle of chemiluminescence resonance energy transfer (CRET). The luminescent dyes excited by CRET emit bright chemiluminescence. The brightness and color of the chemiluminescence emitted in this reaction depend on the properties of the luminescent dyes excited by CRET. Based on the above principles, the integrated immunoassay using chemiluminescence detection of phenylglyoxal derivatives, such as Fig.18 As shown, an antibody coupled with single-stranded DNA (or RNA) and with a luminescent dye is used. Single-stranded DNA (or RNA) must contain 5 to 15 guanines and other components (e.g., adenine, cytosine and thymine in DNA, adenine, cytosine and uracil in RNA), so that a powerful high-energy intermediate can be formed by the reaction of guanine and phenylglyoxal derivatives. In addition, the guanine in the single-stranded DNA (or RNA) should be adjacent to the luminescent dye with the single-stranded DNA (or RNA) label, so as to transfer stronger energy to the luminescent dye from the high-energy intermediate formed by the reaction of polyguanine and phenylglyoxal derivatives. Thus, the single-stranded DNA (20-100bp) is coupled with a luminescent dye and a phenylglyoxal derivative, and the integrated immunoassay method with chemiluminescent detection function is invented. For example, the integrated immunoassay method with chemiluminescent detection, which couples single-stranded DNA (30 bp) with Pacific Blue, TMPG (1-10 mM) in N,N-dimethylformamide (DMF) and tetra-n-butylammonium hydroxide (TPA, 15-30 mM in deionized water), can be used to quantitatively determine trace levels of D-dimer in human serum, and its reaction mechanism is as follows Fig.19 In addition, the integrated immunoassay using chemiluminescent detection of phenylglyoxal derivatives can be used as a multiplex immunoassay tool. For example, three different target antigens present in a sample can be simultaneously quantified using the multiplex integrated immunoassay, which is capable of detecting three different colors of light using a photometer with three photomultiplier tubes to filter and measure specific colors of chemiluminescence, such as Fig. 20 Each antibody with good specific capture ability for the target antigen in the sample is coupled to a different luminescent dye to emit chemiluminescence of a unique color.

[0124] 2.5 Integrated immunoassay with ODI chemiluminescent detection using antibodies coupled to luminescent dyes

[0125] like Fig.14 , 16As shown in Figures 1 and 17, the luminescent dye emits chemiluminescence of a unique color when H2O2 and an ODI derivative are added. Therefore, the integrated immunoassay with ODI chemiluminescent detection is operated using an antibody coupled to the luminescent dye. For example, the integrated immunoassay is based on Fig.21 The reaction mechanism shown in is used to quantify the trace levels of D-dimer in human serum. Human serum samples containing D-dimer are incubated at room temperature for 15 minutes with antibodies coupled to rhodamine 101 through a linker or single-stranded DNA (30-100bp). Then, the rhodamine 101 coupled to the antibody bound to the D-dimer emits red chemiluminescence when H2O2 in isopropanol and ODI in ethyl acetate are added. The distance between the antibody and rhodamine 101 connected by single-stranded DNA is farther than the distance coupled by a linker. Therefore, the integrated immunoassay using the D-dimer antibody coupled to rhodamine 101 through single-stranded DNA is more sensitive than the integrated immunoassay through a linker, because rhodamine 101 under the former condition can emit brighter chemiluminescence without the interference effect of the large-size D-dimer antibody. In addition, the integrated immunoassay with ODI chemiluminescence detection operated using an antibody coupled to a luminescent dye can be used as a method capable of simultaneously quantifying the presence of Fig. 22 Multiplex integrated immunoassays of different target antigens in the samples shown. Fig.21 and 22 It was demonstrated that the integrated immunoassay with ODI chemiluminescent detection can be operated with antibodies coupled to alternative luminescent materials, such as luminescent nanospheres and luminescent carbon dots, instead of small luminescent dyes.

[0126] 3. Integrated immunoassay and colorimetric detection

[0127] 3.1 Integrated immunoassay with colorimetric detection using HRP-conjugated antigen-binding antibodies

[0128] like Figure 23-26As shown, several different types of substrates can be used for the integrated immunoassay with colorimetric detection, and the colorimetric detection uses an HRP-labeled antibody that can specifically capture the target antigen in the sample. Therefore, the sensitivity of the integrated immunoassay with colorimetric detection depends on the properties of the substrate added for the enzyme reaction in the presence of HRP and H2O2. In addition, the sensitivity of the integrated immunoassay depends on the incubation time of the HRP enzyme reaction. However, the incubation time of the integrated immunoassay is much shorter than the incubation time of conventional immunoassays (e.g., competitive and sandwich immunoassays). For example, in the absence of washing, the integrated immunoassay with colorimetric detection is used to quantify the D-dimer in human serum within 30 minutes. However, it is required to incubate and wash for 90 minutes using a commercially available sandwich enzyme-linked immunosorbent assay (ELISA, abcam) using HRP-labeled detection antibodies to quantify it. In addition, the detection limit (1.2ng / ml) of rapid integrated immunoassay is slightly lower than the detection limit of complex and time-consuming ELISA.

[0129] 3.2 ALP-coupled antigen-binding antibody colorimetric detection integrated immunoassay

[0130] Using pNPP (disodium p-nitrophenyl phosphate) as a substrate for ALP-labeled antibodies, the integrated immunoassay with colorimetric detection can be applied to rapidly quantify the target antigen in a sample, such as Fig. 27 As shown. For example, the integrated immunoassay was used to quantitatively determine trace levels of D-dimer in human serum within 30 minutes. The detection limit of the integrated immunoassay using the ALP labeled antibody of pNPP (3.5 ng / ml) was slightly higher than the detection limit of the integrated immunoassay using the HRP labeled antibody of TMB (1.2 ng / ml). However, since the normal range of D-dimer in human serum is as high as 250 ng / ml, the integrated immunoassay using the ALP labeled antibody of pNPP can also be used for the quantification of D-dimer.

[0131] 3.3 Integrated immunoassays with colorimetric detection using antigen-binding antibodies coupled to other enzymes rather than conventional enzymes such as ALP and HRP

[0132] The integrated immunoassay with colorimetric detection can be performed with antibodies coupled to other enzymes instead of HRP and ALP, such as Fig.28 and 29As shown. The sensitivity of the integrated immunoassay with colorimetric detection is determined by the properties of the alternative enzyme (e.g., β-galactosidase, glucose oxidase) rather than the conventional enzyme. In addition, the incubation time of the enzyme reaction depends on the properties of the alternative enzyme. The substrates for the integrated immunoassay using glucose oxidase labeled antibodies for colorimetric detection are the same as those using HRP labeled antibodies (e.g., TMB, ABTS, OPD, Amplex Red).

[0133] 4. Integrated immunoassay with electrochemical detection using an antigen-binding antibody coupled to an enzyme

[0134] like Fig.30 and 31 As shown in FIG. 1 , the integrated immunoassay with electrochemical detection function capable of measuring current is performed by antibodies labeled with conventional enzymes (e.g., ALP, HRP) that bind to target antigens. First, the target antigen interacts with the antibody-coupled ALP (or HRP) weakly immobilized on the surface of the graphene oxide membrane fixed on the pores, as shown in FIG. Figure 2 As shown. After incubation for a certain period of time (10-30 minutes), the solution containing the antigen-bound antibody-coupled ALP (or HRP) is transferred from the well to an electrochemical cell with a glassy carbon electrode. An appropriate substrate capable of reacting with a specific enzyme (such as ALP, HRP) is then added to the electrochemical cell. The final mixture is incubated for a certain period of time (5-20 minutes). After the final incubation, the current generated from the electrochemical cell is measured. The difference between the currents measured in the absence and presence of the target antigen increases with the increase in the concentration of the target antigen. For example, based on Fig.31 The mechanism shown in the present invention is used to quantify trace levels of D-dimer in human serum with good reliability using the integrated immunoassay with electrochemical detection, which uses specific antibodies coupled to HRP, H2O2 and acetaminophen as substrates. The sensitivity and incubation time of the integrated immunoassay with electrochemical detection depend on the type of substrate or electrode used.

[0135] 5. Electrochemiluminescence detection integrated immunoassay

[0136] 5.1 Integrated immunoassay with electrochemiluminescence detection of ruthenium complex-labeled antibody binding to target antigen

[0137] Fig.32 The top image shows the target antigen (Ru(bpy)3) of the antibody-labeled ruthenium complex. 2+ <antibody> antigen) is formed by the integrated immunoassay procedure (see Figure 2 ). Figure 2 The first procedure of the integrated immunoassay shown in FIG. 5 forms a Ru(bpy)3 2+A small volume of <antibody> antigen solution is transferred to the electrochemiluminescent cell. Ru(bpy)3 2+ Oxidation on glassy carbon electrode (GCE) to produce Ru(bpy)3 3+ ,like Fig.32 In addition, tripropylamine, as an electrochemiluminescent co-reagent, is also oxidized to form tripropylamine + on the GCE electrode. Then, Ru(bpy)3 3+ Reacts with tripropylamine + to produce a high-energy intermediate (Ru(bpy)3 2+* ). Finally, by converting the excited state of Ru(bpy)3 2+* To the ground state of Ru(bpy)3 2+ The rapid transition of Ru(bpy)3 produces intense electrochemiluminescence, which is measured at 620 nm using a photometer with a photomultiplier tube (PM tube). 2+ In the integrated immunoassay as a labeling material, the brightness of electrochemiluminescence increases proportionally with the increase of target antigens (e.g., viruses, cells, proteins, peptides, small molecules) in the sample. At the same time, the brightness of electrochemiluminescence in conventional immunoassays increases or decreases with the increase of target antigens, because sandwich immunoassays can quantify large molecular target antigens (e.g., viruses, cells, proteins, peptides), while competitive immunoassays can analyze small molecular target antigens such as triiodothyronine (T3) and thyroxine (T4). For example, using Ru(bpy)3 2+ The complex electrochemiluminescence assay quantifies trace levels of D-dimer in human serum. As the concentration of D-dimer increases, the brightness of the electrochemiluminescence increases proportionally.

[0138] The target antigen bound by the antibody labeled with the nanoparticles (eg, gold (Au), silver (Ag)) formed by the integrated immunoassay can be used to generate the target antigen from Ru(bpy)3 2+ Electrochemiluminescence emission can be achieved because the nanoparticles can act as a co-reagent, such as tripropylamine, in the electrochemiluminescence reaction. For example, the target antigen (Au<antibody>antigen) bound by an antibody labeled with gold nanoparticles is Figure 2 The first step of the integrated immunoassay is shown in FIG. A small volume of solution containing the Au<antibody> antigen is transferred to an electrochemiluminescent cell. Fig.33 As shown, Au-labeled antibody-binding antigen can be used as a 3+ The reaction produces Ru(bpy)3 2+* The role of Au is the same as that of propylamine in the electrochemiluminescence reaction of ruthenium complexes. Finally, in the excited state of Ru(bpy)3 2+* Rapid transition to the ground state of Ru(bpy)3 2+During the measurement of strong electrochemiluminescence, such as Fig.33 Therefore, by Fig.33 The emission wavelength of electrochemiluminescence emitted by the procedure shown is Fig.32 The mechanisms shown produce emission at consistent wavelengths.

[0139] 5.2. Integrated immunoassay using electrochemiluminescence detection using ABEI-labeled antibodies binding to target antigens

[0140] Fig.34 Shown by Figure 2 The ABEI-labeled antibody-bound target antigen (ABEI <antibody> antigen) formed by the first procedure of the integrated immunoassay shown in can also produce strong electrochemiluminescence in an electrochemiluminescent cell. First, a small volume of ABEI <antibody> antigen is transferred to an electrochemiluminescent cell. ABEI 1 interacts with GCE to produce intermediate 2. Then, 2 reacts with H2O2 to produce a high-energy intermediate 3 in an excited state. Finally, a photometer with a PM tube is used to measure the strong electrochemiluminescence generated by the rapid transition from the excited state to the ground state. The brightness of the electrochemiluminescence increases proportionally with the increase of any size of target antigen (e.g., virus, cell, protein, peptide, small molecule) present in the sample. For example, trace levels of D-dimer in quantitative human serum are detected with ABEI chemiluminescence. As the concentration of D-dimer increases, the brightness of the electrochemiluminescence increases proportionally.

[0141] 6. Integrated immunoassay with fluorescence detection

[0142] 6.1. Integrated immunoassay using enzymes and fluorescence detection

[0143] The integrated immunoassay can be operated with a fluorescence detection capable of sensing visible light emitted by a fluorescent dye formed by the reaction of a non-fluorescent substrate (e.g., FDP, Amplex Red) and an enzyme (e.g., ALP, HRP) excited by a light source such as a laser, LED, or xenon lamp. Fig.35 As shown, the first procedure of the integrated immunoassay (see Figure 2 ) is formed by an enzyme-labeled antibody-bound antigen (enzyme <antibody> antigen) reacting with a substrate for a certain incubation time to produce a fluorescent dye. Then, the fluorescent dye excited by a light source emits visible fluorescence within a certain incubation time. The color of the fluorescence depends on the properties of the fluorescent dye. For example, the integrated immunoassay with fluorescence detection operated with HRP, Amplex Red and H2O2 is used to quantify the trace level of D-dimer in human serum. The detection limit of the integrated immunoassay with fluorescence detection capable of quantifying D-dimer within 20 minutes is as low as 0.5ng / mL, which is 500 times lower than the normal D-dimer concentration in human serum.

[0144] 6.2 Integrated immunoassays using fluorescent dye-labeled antibodies for fluorescence detection

[0145] The integrated immunoassay with fluorescence detection can also be applied to fluorescent dye-labeled antibody-bound antigen (fluorescent dye <antibody> antigen). Fig.35 As shown, the first procedure of the integrated immunoassay (see Figure 2 ) formed by the fluorescent <antibody> antigen emits visible fluorescence. The color of the fluorescence emitted in the integrated immunoassay depends on the properties of the fluorescent dye, such as Fig.36 As shown. For example, the integrated immunoassay was used to quantify D-dimer in human serum within 20 minutes, wherein fluorescence detection was operated using fluorescein-labeled antibodies. Even using the same fluorescence detection, the detection limit of the integrated immunoassay using fluorescein-labeled antibodies (8.2 ng / ml) was about 16 times higher than the detection limit of the integrated immunoassay using HRP-labeled antibodies (0.5 ng / ml). This is because the brightness of the fluorescence emitted by the large antibody labeled with a small fluorescent dye is quenched due to the interference of the antibody. However, Fig.36 It was demonstrated that the integrated immunoassay with fluorescence detection operated by fluorescent <antibody> antigen can be used as a multiplex assay method capable of simultaneously sensing different types of target antigens in a sample.

[0146] 7. Integrated immunoassay that can sense positive or negative samples with the naked eye

[0147] The integrated immunoassay can be applied to observe positive or negative results with the naked eye. For example, the color of ZonylFSN-100 functionalized gold nanoparticles (FSN-AuNPs) changes from red to purple in the presence of cysteamine, which is formed by the reaction of ALP-labeled antibody-bound antigen formed by the first procedure of the integrated immunoassay (see Figure 2 ), and cysteamine S-phosphate is a substrate that can react with ALP. As the concentration of the antigen in the sample increases, the intensity of the purple color displayed by the aggregation of FSN-AuNPs increases, because cysteamine supports the aggregation of FSN-AuNPs. Therefore, it can be based on Fig.37 The mechanism shown develops the integrated immunoassay capable of sensing both positive and negative samples with the naked eye.

[0148] 8. Integrated Immunoassay Using a Radiometer

[0149] In conventional radioimmunoassay methods, competitive radioimmunoassay uses isotopes (e.g. 125I) coupled antigen, while sandwich radioimmunoassay uses a detection antibody coupled to an isotope. Both methods require time-consuming multiple washing and incubation steps, as shown in Figures 3 and 4, which are complex procedures for other conventional immunoassays. In addition, the radioactivity measured by competitive radioimmunoassay decays exponentially with the increase of target antigen in the sample, while the radioactivity measured by sandwich radioimmunoassay increases proportionally with the increase of target antigen concentration.

[0150] Fig.38 It is shown that the integrated immunoassay using a radiometer can analyze the target antigen in the sample, and the isotope-labeled antibody-bound antigen (isotope <antibody> antigen) formed in the presence of the target antigen in the sample can be produced without washing and one incubation. After the first procedure of the integrated immunoassay, the solution in the test kit needs to be transferred to the detection well to remove the relatively high background generated by the isotope-labeled antigen fixed on the surface of the test kit after incubation. The radioactivity of the solution is measured with a gamma counter in the absence and presence of the isotope <antibody> antigen. Therefore, the radioactivity of the solution in the detection well increases proportionally with the increase in the antigen concentration in the sample.

[0151] Hereinafter, the preparation of the device for the integrated immunoassay according to an exemplary embodiment of the present disclosure is described in detail by way of examples. However, the following examples are provided only to illustrate the present disclosure, and the scope of the present disclosure is not limited thereto.

[0152] B. Temporary Immobilization of Antibodies on Carbon Nanomaterial Surfaces

[0153] For operation Figure 2 In the integrated immunoassay shown in , it is important to prepare antibodies temporarily attached to the surface of carbon nanomaterials based on the π-π stacking interaction between antibodies and carbon materials. In addition, removing antibodies remaining in the solution after the π-π stacking interaction is a very important factor in reducing the background measured in the absence of target antigens and enhancing the sensitivity in the integrated immunoassay. There are two specific methods for preparing antibodies temporarily fixed to the surface of carbon nanomaterials, which can be applied as shown below.

[0154] Example 1. Integrated immunoassay of antibodies on the surface of magnetic carbon nanomaterials

[0155] 1.1 Preparation of magnetic carbon nanomaterials

[0156] A certain concentration (1-5 mg / mL) of a specific carbon material (e.g., graphene oxide, reduced graphene oxide, graphene, single-walled or multi-walled carbon nanotubes) is mixed with FeCl2 (0.1-0.6 mg / mL) and FeCl3 (0.03-0.2 mg / mL) in deionized water. The mixture is placed in an oven and heated to 85°C. Then, NH4OH (0.4-0.6%) is added to the mixture. The final mixture is shaken and incubated in an oven at 85°C for an appropriate amount of time (45-60 minutes) to convert the carbon material into a magnetic carbon nanomaterial. After incubation, the solution containing the magnetic carbon nanomaterial is transferred to a 1.5 mL centrifuge tube. The magnetic carbon nanomaterial is then washed 3-4 times with deionized water using a magnetic separator. The pure magnetic carbon material is stored in deionized water under ambient conditions. As Fig.39 As shown in (a)-39(c), the surface of the magnetic carbon nanomaterial (e.g., magnetic graphene oxide containing iron) is different from the surface of pure graphene oxide. In addition, Fig.39 (d) shows that the antibodies attached to the magnetic graphene oxide can be separated from the solution containing free antibodies remaining after the π-π stacking interaction attaches the antibodies to the magnetic graphene oxide.

[0157] 1.2. Preparation of antibodies temporarily immobilized on the surfaces of six different magnetic carbon nanomaterials

[0158] Using the above method, six different types of magnetic carbon nanomaterials were prepared using graphene oxide, reduced graphene oxide, graphite, 10-20nm, 20-30nm and 30-50nm multi-walled carbon nanotubes as raw materials and stored at room temperature.

[0159] By adding acridine NHS ester that can be combined with the primary amine group of antibody, prepare the specific antibody coupled with acridine ester in PBS. The mixture is incubated at room temperature for 30 minutes, and the free antibody remaining after the reaction is removed by centrifugal filter tube (3, 10 or 30MWCO) for 2-3 times. The acridine ester (1mg / mL) coupled with the antibody in PBS is stored in a refrigerator.

[0160] The antibody-conjugated acridinium ester (1 μg / mL) diluted with the stock solution was mixed with the magnetic carbon nanomaterial (20 μg / mL) in PBS. The mixture was inserted into a rotor (18 rpm) and incubated at room temperature for 30 minutes. After the reaction, the antibody-conjugated acridinium ester immobilized on the surface of the magnetic carbon nanomaterial was separated and washed with a magnetic separator. Fig.40 Shown is the separation of six different magnetic carbon nanomaterials bound to an acridinium ester conjugated to an antibody.

[0161] 1.3. Quantification of D-dimer by the integrated immunoassay using antibodies attached to the surface of magnetic carbon nanomaterials

[0162] The antibody-coupled acridinium ester immobilized on the magnetic carbon nanomaterial (20 μg / mL) (in 100 μl PBS in a 1.5 mL centrifuge tube) was inserted into a magnetic separator. Then, the clarified PBS solution was removed from the 1.5 ml centrifuge tube. Various concentrations of D-dimer in human serum were prepared as standards for the integrated immunoassay.

[0163] A standard containing a certain concentration of D-dimer (100 μl) was added to a 1.5 mL centrifuge tube containing the antibody-conjugated acridinium ester immobilized on the magnetic carbon nanomaterial. The mixture was incubated for 15 min while rotating in a rotor (18 rpm).

[0164] After incubation, the solution containing the D-dimer antigen-bound antibody-conjugated acridinium ester was removed from the 1.5 mL centrifuge tube using a magnetic separator.

[0165] In order to measure the chemiluminescence emitted from the standard, 25 μl of the standard was placed in a borosilicate test tube. Then, the test tube was inserted into the detection cell of the photometer. After clicking the start button of the photometer equipped with two syringes, 20 mM of 0.1 N HNO3 (25 μl) was injected into the test tube by the first syringe pump. After a 3 second interval, 0.25 M NaOH containing Triton X-100 (125 μl) was injected into the test tube by the second syringe pump to immediately measure the chemiluminescence for 2 seconds.

[0166] As the concentration of D-dimer increases, the relative chemiluminescence intensity increases proportionally. However, the sensitivity of the integrated immunoassay depends on the properties of the magnetic carbon nanomaterial used. For example, Fig.41 It is shown that the integrated immunoassay operated with magnetic graphene oxide is more sensitive than the integrated immunoassay operated with other magnetic carbon nanomaterials. This is because the CL / CLO ratio determined by the integrated immunoassay method with magnetic graphene oxide is greater than the CL / CLO ratio calculated with other magnetic carbon nanomaterials. CL is the chemiluminescence intensity measured in the presence of 2.5 ng / mL D-dimer, while CLO is the background measured in the absence of D-dimer. Fig.39 It is shown that the integrated immunoassay operated with magnetic graphene oxide can quantify D-dimer in human serum as low as 2.5 ng / ml within a statistically acceptable error range (<5%). The normal range (cutoff value) of D-dimer in human serum is 250 ng / ml in human serum. Therefore, the integrated immunoassay operated with magnetic graphene oxide can quantify D-dimer in human serum with good reliability. Fig.41As shown, the CL / CL0 in the presence of other magnetic carbon nanomaterials is slightly higher than 1. These results indicate that the π-π stacking strength between the antibody and magnetic graphene oxide is the weakest. Therefore, the binding interaction between D-dimer and antibody occurs on the surface of magnetic graphene oxide.

[0167] Example 2. Integrated immunoassay with antibodies temporarily immobilized on the surface of graphene oxide membrane

[0168] 2.1 Preparation of carbon nanomaterial film on polystyrene pore surface

[0169] 140 μl of carbon nanomaterial (2 mg / mL) dispersed in aqueous Tris-HCl (10 mM, pH 8.5) was inserted into the polystyrene wells of a 96-well plate. Six different carbon nanomaterials, such as graphene oxide, reduced graphene oxide, graphene, 10-20 nm multi-walled carbon nanotubes, 20-30 nm multi-walled carbon nanotubes, and 30-50 nm multi-walled carbon nanotubes were used to prepare carbon nanomaterial films fixed on the flat polystyrene well surface.

[0170] The polystyrene wells containing one of the six different types of carbon nanomaterials were incubated in an oven at room temperature or 65°C to form a carbon nanomaterial film by water evaporation.

[0171] like Fig.42 As shown in (a), the graphene oxide membrane was prepared by uniformly dispersing graphene oxide particles. The graphene oxide membrane was stable for at least 24 h in the presence of buffer or human serum. However, the reduced graphene oxide and graphene membrane on the polystyrene pore surface were broken apart due to the aggregation of nanoparticles, as shown in Fig.42 (b) As shown. The results show that it is difficult to apply reduced graphene oxide and graphene membrane in the integrated immunoassay. This is because the accuracy of the integrated immunoassay using split reduced graphene oxide and graphene membrane is not as good as the accuracy of the integrated immunoassay using a uniform graphene oxide membrane. Unfortunately, no multi-walled carbon nanotube membrane was produced. Fig.42 As shown in (c), even though the dried MWCNTs on the polystyrene pores looked like a uniform film or had split, the MWCNTs were immediately dispersed upon the addition of PBS.

[0172] 2.2 Graphene oxide film fixed on polystyrene surface

[0173] The strong graphene oxide film on the polystyrene surface was produced by evaporating water from the buffer used to disperse the graphene oxide nanoparticles in the polystyrene pores. The production of a uniform graphene oxide film on the surface depends on the components present in the specific buffer solution, pH (6.5-9) and temperature (20-65°C) to promote the evaporation of water in the buffer.

[0174] For example, Fig.43 It is shown that uniform graphene oxide films are prepared by dispersing graphene oxide nanoparticles in phosphate buffer (pH 7), PBS (pH 7.4), and Tris-HCl (pH 8, 8.5, and 9). However, the graphene oxide films formed in Tris-HCl (pH 7 and 7.5) split irregularly. The results show that the production of strong and uniform graphene oxide films depends on the nature of the components present in the buffer and mixed with the graphene oxide nanoparticles, and the process of evaporation of water in the buffer at a certain temperature (20-70°C). Fig.43 (a) shows that a uniform graphene oxide film is formed in PBS (pH 7.4), while an irregular and split graphene oxide film is produced in Tris buffered saline (TBS) (pH 7.4). Even though the graphene oxide film will split in phosphate buffer (pH 6), a uniform graphene oxide film can be produced using graphene oxide nanoparticles in phosphate buffer (pH 6.5 or higher). The results show that the main component for producing a uniform graphene oxide film in PBS is phosphate ions. NaCl and KCl present in PBS and TBS are not the main factors for producing a uniform graphene oxide film.

[0175] 2.3. Integrated immunoassays operated with graphene oxide membranes formed in various buffers

[0176] Add 70 μL of antibody conjugated with acridinium ester (1 μg / mL) in PBS to Fig.43 The antibody was then incubated at room temperature for 30 minutes to temporarily immobilize the antibody on the surface of each graphene oxide film using the π-π stacking interaction between the antibody and the graphene oxide film. After incubation and washing with PBS, antibody-conjugated acridinium esters weakly bound to each graphene oxide film were generated.

[0177] Prepare a standard without D-dimer to measure background (CL0) in the integrated immunoassay. Prepare another standard containing D-dimer (2.5ng / mL) to measure the chemiluminescence (CL) emitted in the integrated immunoassay. Each standard (80μL) is added to each hole containing acridinium esters of antibody couplings weakly bound on graphene oxide film. The mixture is incubated at room temperature for 15 minutes. After incubation, 25μl of each standard containing or not containing acridinium esters of antibody couplings of D-dimer antigen binding is added to a borosilicate test tube. The test tube is inserted into the detection cell of a photometer with two injection pumps. Then, 20mM H2O2 in 0.1N HNO3 (25μl) is injected into the test tube using the first injection pump. Then, after adding 0.25M NaOH containing Triton X-100 (125μl) using the second injection pump, the chemiluminescence emitted from each test tube is measured immediately for 2 seconds.

[0178] like Fig.44 As shown, CL / CL0 depends on the properties of each graphene oxide film. In addition, Fig.44 The results shown in show that the integrated immunoassay operated with all uniform graphene oxide membranes formed in Tris-HCl (pH 8-9), PBS (pH 7.4) and sodium phosphate (pH 7) can be applied to rapidly quantify trace levels of D-dimer in human serum. The integrated immunoassay operated with graphene oxide membranes formed in sodium phosphate buffer (pH 7) is more sensitive than other integrated immunoassays produced with graphene oxide membranes in Tris-HCl buffer (pH 7-9), TBS (pH 7.4) and PBS (pH 7.4). Fig.43 As shown in Figure 2, the sensitivity of the integrated immunoassay using irregularly split graphene oxide membranes formed in Tris-HCl (pH 7 and 7.5) and TBS (pH 7.4) is not as good as that using uniform graphene oxide membranes. In addition, the sensitivity of the integrated immunoassay operated with uniform graphene oxide membranes formed in Tris-HCl buffer depends on pH. Fig.45 As shown in Figure 5, the integrated immunoassays with uniform graphene oxide membranes formed in Tris-HCl (pH 8) were more sensitive than those formed in Tris-HCl (pH 8.5 and 9). In addition, the sensitivity of the integrated immunoassays operated with uniform graphene oxide membranes depended on the pH of the sodium phosphate buffer. Fig.45 As shown, the integrated immunoassay using the graphene oxide membrane formed in sodium phosphate buffer (pH 7.5) is more sensitive than the rest of the integrated immunoassays using graphene oxide membranes produced in other sodium phosphate buffers. Fig.45As shown, the sensitivity of the integrated immunoassay with the irregularly split graphene oxide membrane formed in sodium phosphate buffer (pH 6) is not as good as that of the integrated immunoassay with the uniform graphene oxide membrane.

[0179] 2.4 Role of the buffer containing specific antibodies in the integrated immunoassay

[0180] The sensitivity of the integrated immunoassay depends on the buffer containing antibodies that can weakly bind to the uniform graphene oxide film. The integrated immunoassay using PBS (pH 7.4) containing D-dimer antibodies coupled to acridinium esters is more sensitive than the rest of the integrated immunoassays using other buffer solutions containing the same antibodies, such as Fig.46 In addition, the CL / CL0 of the integrated immunoassay using PBS or TBS (pH 7.4) containing D-dimer antibodies coupled to acridinium esters was higher than those using Tris-HCl and sodium phosphate buffers containing the same antibodies. Fig.46 It was shown that NaCl and KCl can enhance the sensitivity of the integrated immunoassay.However, all of the integrated immunoassays using various buffers containing antibodies and uniform graphene oxide membranes can be used to quantify trace levels of D-dimer in human serum.

[0181] 2.5 Determination of CL / CL using the integrated immunoassay with bioluminescence, chemiluminescence or electrochemiluminescence detection

[0182] 70 μL of D-dimer antibody coupled with luciferase (0.25 μg / mL) in PBS (pH 7.4) was added to the surface of the graphene oxide membrane fixed in the polystyrene well. Then, it was incubated at room temperature for 30 minutes. The PBS solution containing D-dimer antibody coupled with luciferase remaining after incubation was removed. Then, the wells were washed 3 times with PBS. The luciferase-coupled D-dimer antibody weakly fixed on the surface of the graphene oxide membrane was used to detect the presence of luciferase-coupled D-dimer antibody using a luciferase-based Figure 7 The procedure shown in the figure is an integrated enzyme immunoassay with bioluminescent detection to quantify D-dimer in human serum.

[0183] 70 μL of D-dimer antibody coupled with acridinium ester (1 μg / mL) in PBS (pH 7.4) was added to the surface of the graphene oxide membrane fixed in the polystyrene well. Then, it was incubated at room temperature for 30 minutes. The PBS solution containing D-dimer antibody coupled with acridinium ester remaining after incubation was removed. Then, the well was washed once with PBS. The D-dimer antibody coupled with acridinium ester weakly fixed on the surface of the graphene oxide membrane was used for the detection of the D-dimer antibody using a PCR-based Fig.10The procedure shown in (a) is the integrated immunoassay with acridine chemiluminescent detection to quantify D-dimer in human serum.

[0184] 70 μl of D-dimer antibody coupled with horseradish peroxidase (HRP, 0.1 μg / mL) in PBS (pH 7.4) was added to the surface of the graphene oxide membrane fixed in the polystyrene well. Then, it was incubated at room temperature for 30 minutes. The PBS solution containing the D-dimer antibody coupled with HRP remaining after incubation was removed. Then, the wells were washed 4 times with PBS. The HRP-coupled D-dimer antibody weakly fixed on the surface of the graphene oxide membrane was used for the detection of the D-dimer antibody using a PCR-based Fig.12 The integrated enzyme immunoassay with luminol detection according to the procedure shown in is used to quantify D-dimer in human serum. In addition, the integrated enzyme immunoassay with ODI chemiluminescent detection is operated using D-dimer antibodies coupled to HRP weakly immobilized on the surface of graphene oxide membranes for quantification of D-dimer in human serum, as shown in Fig.14 as shown in .

[0185] 70 μL of D-dimer antibody coupled with alkaline phosphatase (ALP, 0.13 μg / mL) in PBS (pH 7.4) was added to the surface of the graphene oxide membrane fixed in the polystyrene well. Then, it was incubated at room temperature for 30 minutes. The PBS solution containing D-dimer antibody coupled with ALP remaining after incubation was removed. The wells were then washed 3 times with PBS. The ALP-coupled D-dimer antibody weakly fixed on the surface of the graphene oxide membrane was used to detect the presence of D-dimer antibody using a 50 μL PBS-based Fig.15 The integrated enzyme immunoassay with AMPPD chemiluminescent detection according to the procedure shown in is used to quantify D-dimer in human serum. In addition, the integrated enzyme immunoassay with ODI chemiluminescent detection is performed using D-dimer antibodies coupled to ALP weakly immobilized on the surface of graphene oxide membranes for quantification of D-dimer in human serum, as shown in Fig.16 as shown in .

[0186] 70 μL of 6-FAM (1 μg / mL)-coupled D-dimer antibody bound to single-stranded DNA in PBS (pH 7.4) was added to the surface of the graphene oxide membrane fixed in the polystyrene well. Then, it was incubated at room temperature for 30 minutes. The PBS solution containing 6-FAM-coupled D-dimer antibody bound to single-stranded DNA remaining after incubation was removed. Then, the well was washed once with PBS. The 6-FAM-coupled D-dimer antibody bound to single-stranded DNA weakly fixed on the surface of the graphene oxide membrane was used to detect the presence of 6-FAM-coupled D-dimer antibody based on the PBS solution. Fig.19The procedure shown in the figure is an integrated immunoassay with phenylglyoxal derivative detection to quantify D-dimer in human serum.

[0187] Mix 70 μL of PBS (pH 7.4) with Ru(bpy)3 2+ The D-dimer antibody (1 μg / mL) coupled to the graphene oxide membrane was added to the surface of the graphene oxide membrane fixed in the polystyrene pores. Then, it was incubated at room temperature for 30 minutes. The remaining Ru(bpy)3 2+ PBS solution of the coupled D-dimer antibody. Then, the wells were washed twice with PBS. Ru(bpy)3 weakly immobilized on the surface of the graphene oxide membrane 2+ Conjugated D-dimer antibodies are used to Fig.32 The procedure shown in the figure is an integrated immunoassay for electrochemiluminescence detection to quantify D-dimer in human serum.

[0188] The sensitivity of the integrated immunoassay depends on the nature of the material coupled to the D-dimer antibody and the detection method. Fig.47 It was shown that each of the integrated immunoassays with specific detection such as bioluminescence, chemiluminescence or electrochemiluminescence can rapidly quantify 2.0 ng / mL D-dimer in human serum with good accuracy. The results show that any of the integrated immunoassays with detection can be used to rapidly monitor trace levels of D-dimer in human serum.

[0189] 2.6 Correlation study between integrated immunoassay and chemiluminescence detection

[0190] A total of eight human serum standards containing different concentrations of troponin I (e.g., 0, 8, 16, 31, 63, 125, 250, 500 pg / ml) were prepared. These standards were used to obtain linear calibration curves for the three integrated immunoassays using different chemiluminescent detection methods (e.g., acridine, luminol, and AMPPD chemiluminescence). In addition, 12 unknown human serum samples were used to study the correlation between the three different integrated immunoassays.

[0191] 80 μL of standard (or unknown sample) is placed in a polystyrene well prepared for the integrated immunoassay with acridine chemiluminescent detection. The mixture is incubated at room temperature for 10 minutes. After incubation, 25 μL of a solution containing (or not containing) TnI antigen-bound antibody-coupled acridine ester is transferred to a borosilicate test tube. The test tube is inserted into the detection cell of a photometer with two syringe pumps. Using the first syringe pump, 20mM H2O2 in 0.1N HNO3 (25 μL) is injected into the test tube. After 3 seconds, the second syringe pump injects 0.25M NaOH containing Triton X-100 into the test tube to measure the chemiluminescent intensity for 2 seconds. As the concentration of TnI increases, the relative chemiluminescent intensity increases proportionally.

[0192] 80 μL standards (or unknown samples) are placed in the polystyrene wells of the integrated immunoassay prepared for detection using luminol chemiluminescence. The mixture is incubated at room temperature for 8 minutes. After incubation, 25 μL of the solution from the polystyrene wells is transferred to a borosilicate test tube. The test tube is inserted into the detection pool of the photometer with two syringe pumps. The first syringe pump injects a mixture (25 μL) of Tris-HCl (50mM, pH 8.5) containing 7.5mM H2O2 and 1mM 4-iodophenol into the test tube. Then, 0.25mM luminol (25 μL, in Tris-HCl (50mM, pH8.5)) is injected into the test tube by the second syringe pump. The final mixture is incubated for 2 minutes, and then the luminol chemiluminescence emitted from the test tube is measured for 1 second. As TnI concentration increases, relative chemiluminescence intensity is proportionally enhanced.

[0193] 80 μL of standard (or unknown sample) is placed in a polystyrene well prepared for the integrated immunoassay with AMPPD chemiluminescent detection. The mixture is incubated at room temperature for 10 minutes. After incubation, 10 μL of the solution from the polystyrene well is transferred to a borosilicate test tube. The test tube is inserted into the detection cell of a photometer with two syringe pumps. The first syringe pump injects a commercially available AMPPD working solution (25 μL) into the test tube. The final mixture is incubated for 3 minutes, and then the AMPPD chemiluminescence emitted from the test tube is measured for 5 seconds. As the concentration of TnI increases, the relative chemiluminescent intensity increases proportionally.

[0194] like Fig.48 As shown, the analysis results of unknown samples using the integrated immunoassay with chemiluminescent detection were consistent with those using 2 other chemiluminescent detections. Fig.48 The good correlation shown in indicates that the integrated immunoassay can be used to rapidly quantify TnI in human serum using any chemiluminescent detection.

[0195] 2.7 Selectivity of the integrated immunoassay

[0196] The selectivity of the integrated immunoassay depends on the specificity of the antibody that can bind to the target antigen in the sample. In other words, the selectivity of the integrated immunoassay is not determined by detection methods such as bioluminescence, chemiluminescence, colorimetry, electrochemistry, electrochemiluminescence, fluorescence, naked eye, and radiometric determination. For example, the selectivity of the integrated immunoassay with acridine chemiluminescence designed for quantification of TnI in human serum is the same as the selectivity of other integrated immunoassays with chemiluminescence detection (e.g., luminol, ODI, AMPPD, and phenylglyoxal derivative chemiluminescence).

[0197] C. Integrated immunoassay device (kit)

[0198] The components of the integrated immunoassay are synthesized by (1) reacting with a bioluminescent emitter (e.g., a fluorescein derivative), a chemiluminescent emitter (e.g., ABEI, an acridinium ester derivative, a luminescent dye, a single-stranded DNA conjugated with a luminescent dye), an electrochemiluminescent emitter (e.g., ABEI, isoluminol, luminol, Ru(bpy)3 2+ ), enzymes (e.g., ALP, β-galactosidase, glucose oxidase, HRP, luciferase), nanomaterials (e.g., gold, silver nanoparticles), and radioisotopes (e.g., 125 The invention relates to a method for determining the immunoassay by (1) a specific antibody coupled to a biological material or a chemical material and (2) a detection method such as bioluminescence, chemiluminescence, colorimetry, electrochemistry, electrochemiluminescence, fluorescence, naked eye and radiometry. The integrated immunoassay kit has magnetic graphene oxide nanoparticles or a uniform graphene oxide film immobilized on the surface of a polystyrene hole, which can weakly capture the specific antibody coupled to the biological material or the chemical material.

[0199] Although exemplary embodiments of the present disclosure have been described with reference to the accompanying drawings, experts or ordinary technicians in the field will understand that various modifications and changes may be made to the present disclosure without departing from the spirit and scope of the present disclosure defined by the appended claims.

[0200] Therefore, the technical scope of the present disclosure should not be limited by the contents described in the detailed description of the specification but should be defined by the claims.

Claims

1. A device for detecting a target antigen in a sample, comprising: container; Carbon nanomaterials; as well as Antibodies conjugated to markers, wherein the antibody is immobilized on the surface of the carbon nanomaterial, wherein the antibody has a binding site for the target antigen, and When the target antigen binds to the binding site of the antibody, the antibody coupled to the marker is separated from the carbon nanomaterial, and the antibody labeled with the marker that binds to the target antigen generates a signal.

2. The device according to claim 1, wherein the carbon nanomaterial is selected from one or more of graphene, graphene oxide, reduced graphene oxide, carbon nanotubes and carbon nanotube oxides. 3 . The device according to claim 1 , wherein the antibody is immobilized on the surface of the carbon nanomaterial through non-covalent π-π stacking interaction, and the distance between the antibody and the carbon nanomaterial is 10 nm or less.

4. The device of claim 1, wherein the sample is a biological solution selected from the group consisting of serum, plasma, whole blood, sweat, urine, and cerebrospinal fluid.

5. The device of claim 1, wherein the signal generated by the marker is selected from the group consisting of bioluminescence, chemiluminescence, fluorescence, colorimetry, electrochemistry, electrochemiluminescence, radiometry, and visible light.

6. The device according to claim 1, wherein the marker is a bioluminescent marker selected from the group consisting of luciferase, luciferin and luciferin derivatives.

7. The device according to claim 1, wherein the label is selected from the group consisting of acridinium ester chemiluminescent detection, chemiluminescent detection using horseradish peroxidase (HRP) labeled antibodies, chemiluminescent detection using alkaline phosphatase (ALP) labeled antibodies, phenylglyoxal derivative chemiluminescent detection, and chemiluminescent detection labels of ODI chemiluminescent detection operated with antibodies coupled to luminescent dyes.

8. The device of claim 1, wherein the label is selected from the group consisting of colorimetric detection labels for detection: colorimetric detection with an antigen-binding antibody coupled to horseradish peroxidase (HRP), colorimetric detection with an antigen-binding antibody coupled to alkaline phosphatase (ALP), colorimetric detection with an antigen-binding antibody coupled to β-galactosidase, and colorimetric detection with an antigen-binding antibody coupled to glucose oxidase.

9. The device according to claim 1, wherein the label is selected from ruthenium complex (Ru(bpy)3 2+ ), gold nanoparticles, platinum nanoparticles, silver nanoparticles and electrochemiluminescent detection markers of N-(4-aminobutyl)-N-ethylisoluminol (ABEI).

10. The device of claim 1, wherein the label is selected from the group consisting of electrochemically detectable labels of horseradish peroxidase (HRP), alkaline phosphatase (ALP), glucose oxidase, and β-galactosidase.

11. The device according to claim 1, wherein the label is a fluorescent detection label selected from horseradish peroxidase (HRP), alkaline phosphatase (ALP) and fluorescent dye.

12. The device according to claim 1, wherein the label is selected from the group consisting of horseradish peroxidase (HRP) and alkaline phosphatase (ALP) detectable labels by naked eyes.

13. The device according to claim 1, wherein the marker is 125 Radioactive detection marker of I.

14. A method of manufacturing the device of claim 1, comprising: fixing the carbon nanomaterial on the inner surface of the container; providing the antibody coupled to the label; as well as The antibody coupled to the label is brought into contact with the surface of the carbon nanomaterial. 15 . The method according to claim 14 , wherein the carbon nanomaterial is magnetic graphene, graphene oxide, reduced graphene oxide, single-walled carbon nanotube or multi-walled carbon nanotube.

16. The method according to claim 14, wherein the carbon nanomaterial has a thin film form composed of graphene oxide, and the container is a polystyrene container.

17. A method for detecting a target antigen, comprising: Introducing a sample solution into the device according to claim 1; as well as The signal generated by the antibody labeled with the marker bound to the target antigen is detected to detect the target antigen in the sample solution.

18. The method according to claim 17, wherein the carbon nanomaterial is selected from one or more of graphene, graphene oxide, reduced graphene oxide, single-walled carbon nanotubes and multi-walled carbon nanotubes.

19. The method according to claim 17, wherein the carbon nanomaterial has a thin film form composed of graphene oxide, and the container is a polystyrene container. 20 . The method of claim 17 , wherein the antibody is immobilized on the surface of the carbon nanomaterial through non-covalent π-π stacking interactions, and the distance between the antibody and the carbon nanomaterial is 10 nm or less.

21. The method of claim 17, wherein the sample solution is a biological solution selected from the group consisting of serum, plasma, whole blood, sweat, urine, and cerebrospinal fluid.

22. The method of claim 17, wherein the signal generated by the marker-labeled antibody bound to the target antigen is selected from the group consisting of bioluminescence, chemiluminescence, fluorescence, colorimetry, electrochemistry, electrochemiluminescence, radiometry, and visible light.

23. The method of claim 17, wherein the method does not use an artificially produced antigen coupled to the marker.

24. The method of claim 17, wherein the method does not use a detection antibody coupled to the label.

25. The method of claim 17, wherein the antibodies comprise two or more types of antibodies.

26. The method of claim 17, wherein the method does not include a washing procedure to remove waste after introducing the sample solution into the device.

27. The method of claim 17, wherein the signal is emitted within 30 minutes after the sample solution containing the target antigen is introduced into the device.

28. A method for quantifying a target antigen, comprising: Introducing a sample solution into the device according to claim 1; as well as The intensity of the signal is measured to quantify the target antigen in the sample solution.

29. The method according to claim 28, wherein the carbon nanomaterial is selected from one or more of graphene, graphene oxide, reduced graphene oxide, single-walled carbon nanotubes and multi-walled carbon nanotubes.

30. The method of claim 28, wherein the antibody is immobilized on the surface of the carbon nanomaterial through non-covalent π-π stacking interactions, and the distance between the antibody and the carbon nanomaterial is 10 nm or less.

31. The method of claim 28, wherein the sample solution is selected from the group consisting of serum, plasma, whole blood, sweat, urine, and cerebrospinal fluid.

32. The method of claim 28, wherein the signal generated by the marker-labeled antibody bound to the target antigen is selected from the group consisting of bioluminescence, chemiluminescence, fluorescence, colorimetry, electrochemistry, electrochemiluminescence, radiometry, and visible light.

33. The method of claim 28, wherein the method does not use an artificially produced antigen coupled to the marker.

34. The method of claim 28, wherein the method does not use a detection antibody coupled to the label.

35. The method of claim 28, wherein the antibodies comprise two or more types of antibodies.

36. The method of claim 28, wherein the method does not include a washing procedure to remove waste after introducing the sample solution into the device.

37. The method of claim 28, wherein when the concentration of the target antigen in the sample solution increases, the intensity of the signal increases proportionally.

38. The method of claim 28, wherein the signal is generated within 30 minutes after introducing the sample solution into the device.

Images