Multicolor fluorescent liquid phase detection system and use method thereof

Abstract

The invention discloses a multicolor fluorescent liquid phase detection system and a use method thereof, and relates to the technical field of in-vitro diagnosis instruments. The multicolor fluorescent liquid phase detection system comprises a purification unit and a detection unit which are communicated, the detection unit is arranged around the purification unit, the detection unit comprises a plurality of reaction chambers, the side wall of each reaction chamber is made of a light-permeable material treated by Protein A or Protein G, and the side wall of each reaction chamber is coated with an antibody carrying a marker. According to the multicolor fluorescent liquid phase detection system and the use method thereof, after the antibody carrying the marker and the target object of the corresponding to-be-detected sample are subjected to specific reaction, different color signals are presented, and high-sensitivity and multi-target visual detection is realized. By means of the high-specificity antibody fixing capacity of Protein A / G, various different target objects can be detected at the same time, and the method has the advantages of being easy to operate and comprehensive in detection.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnostic devices, and particularly to a multi-color fluorescence liquid-phase detection system and a method for using the same. Background Art

[0002] Protein A is a protein extracted from the cell wall of Staphylococcus aureus, with a molecular weight of 42 kD. It has a stable structure, no disulfide bonds inside the molecule, is sensitive to the acid-base environment, and has the ability to specifically bind to antibodies. Protein A contains 5 highly homologous IgG-binding domains, can form strong affinity with IgG molecules from various sources, and its binding site is located in the Fc segment of IgG, which will not affect the activity of IgG binding to antigens. Therefore, Protein A has important application values in the fields of antibody purification, immunoassay, and biosensing.

[0003] Protein G is a cell wall protein isolated from group G streptococcus, with a molecular weight of 25 kDa. It has an albumin-binding domain at the N-terminus and IgG-binding and cell wall-binding domains at the C-terminus. Protein G can bind to the Fc region of IgG of most mammals and also has a good binding ability to IgG of uncommon species. It is commonly used in the fields of antibody purification, immunoassay, etc.

[0004] Considering portability and visualization of detection results, most current detection devices support single target detection or simultaneous detection of two targets, which have many limitations in actual clinical applications. If more targets need to be detected, multiple test devices usually need to be combined, which not only increases the complexity of operation, but also brings problems such as repeated sampling and different issuing times of individual detection results, thereby limiting doctors' timely and accurate judgment of patients' conditions and delaying the treatment opportunity. To facilitate more timely, comprehensive, and accurate judgment of the condition by clinicians, reduce the detection cost, and make the operation simpler, it is of great significance to develop a multi-color fluorescence liquid-phase detection system for realizing multi-target synchronous detection on a single detection platform. Summary of the Invention

[0005] The present invention provides a multi-color fluorescence liquid-phase detection system and a method for using the same to solve the limitations and cost defects in the prior art. The multi-color fluorescence liquid-phase detection system is an integrated detection and purification system based on Protein A / G treatment, integrating sample purification and multi-target synchronous detection. Only by adding a test sample into the sample addition hole, multiple different targets can be detected simultaneously. Compared with individual detection results, it is more comprehensive, the operation is more convenient, and the detection accuracy is higher, providing strong technical support for precision medicine, individualized treatment, and early disease screening, and well solving the current limitations in detection applications and preparation costs.

[0006] Specifically, it includes the following technical solutions:

[0007] In a first aspect, a multi-color fluorescence liquid-phase detection system is provided, including a purification unit and a detection unit that are connected. The detection unit is arranged around the purification unit. The detection unit includes a plurality of reaction chambers, and the side walls of each reaction chamber are made of light-transmitting materials treated with Protein A or Protein G. Antibodies carrying markers are coated on the side walls of the reaction chambers.

[0008] Preferably, the reaction chambers are not connected to each other.

[0009] Among them, the purification unit is used to remove impurities that interfere with the detection in the sample to be tested.

[0010] Preferably, the overall structure of the multi-color fluorescence liquid-phase detection system is one of a cuboid, a cube, a polyhedron, or a cylinder, and the purification unit is one of a cuboid, a cube, a polyhedron, or a cylinder.

[0011] Among them, Protein A / G (Protein A or Protein G) can specifically bind to the antibody carrying the marker, fix it in the optimal orientation, thereby significantly enhancing the antigen capture ability. Compared with the traditional fluorescence immunochromatography technology, the detection sensitivity and specificity are improved. In addition, the traditional ELISA (ELISA: enzyme-linked immunosorbent assay) needs to go through cumbersome steps such as manual sample addition, incubation, and washing, and the detection process is time-consuming. However, the multi-color fluorescence liquid-phase detection system of the present invention only needs to add samples to achieve full-automatic detection, greatly improving the detection efficiency and convenience.

[0012] Furthermore, the marker is one or more of fluorescent particles, enzymes, chemiluminescent substances, nanomaterials, magnetic microparticles, and rare earth ions.

[0013] Furthermore, the antibody is one or more of SARS-CoV-2 antibody (SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus 2), Flu A antibody (Flu A: Influenza A virus), Flu B antibody (Flu B: Influenza B virus), and RSV antibody (RSV: Respiratory Syncytial Virus).

[0014] Furthermore, the light-transmitting material is one or more of glass, polycarbonate, polymethyl methacrylate, cycloolefin copolymer, and polydimethylsiloxane.

[0015] Among them, the light-transmitting material can directly observe the color change of the detection result, which is convenient for quickly reading data and improving the convenience and visualization degree of detection.

[0016] Further, the marker is a nanomaterial, and the nanomaterial is selected from one of carbon nanoparticles, non-metallic nanoparticles, nano metal particles, latex microspheres, and quantum dots.

[0017] Preferably, the carbon nanoparticles are selected from one or more of carbon nanotubes, carbon nanofibers, and nano carbon spheres.

[0018] Preferably, the non-metallic nanoparticles are colloidal selenium.

[0019] Preferably, the nano metal particles are selected from one or more of nano gold particles, nano silver particles, nano nickel particles, and nano palladium particles.

[0020] Preferably, the latex microspheres are polystyrene latex microspheres modified by chemical groups, and the chemical groups are selected from one of carboxyl group, amino group, hydroxyl group, mercapto group, and sulfate group.

[0021] Preferably, the quantum dots are selected from one or more of elemental semiconductor quantum dots, compound semiconductor quantum dots, alloy quantum dots, core-shell structure quantum dots, and doped quantum dots.

[0022] Preferably, the marker is a fluorescent particle, and the fluorescent particle is selected from one of anthocyanin series fluorescent dyes, carboxyfluorescein, fluorescein isothiocyanate, 2-methoxy fluorescein, rhodamine, phycoerythrin, and lanthanide chelates.

[0023] Preferably, the marker is an enzyme, and the enzyme is selected from one of horseradish peroxidase, alkaline phosphatase, and luciferase.

[0024] Preferably, the marker is a chemiluminescent substance, and the chemiluminescent substance is selected from one of acridinium ester, isoluminol, and ruthenium (II) tris(bipyridine).

[0025] Preferably, the marker is a magnetic particle, and the magnetic particle is selected from a magnetic metal and / or a magnetic metal oxide and a composite formed by surface modification with an organic or inorganic material.

[0026] Among them, the magnetic metal oxide is selected from one or more of Fe, Co, Ni and their corresponding oxides and alloys.

[0027] Preferably, the marker is a rare earth ion, and the rare earth ion is selected from one of scandium ion, yttrium ion, and cerium ion.

[0028] Further, the antibody carrying the marker is used to specifically bind to the target in the sample to be tested and then show a corresponding color signal.

[0029] Among them, by using different antibodies carrying different color markers, corresponding color signals are shown after specific binding to the target in the sample to be detected, thereby realizing highly sensitive and multi-target visual detection.

[0030] Furthermore, the sample to be detected includes one or more of serum, plasma, whole blood, peripheral blood, urine, saliva, fecal extract, oral and human tissue extract.

[0031] Furthermore, the purification unit includes a sample loading hole, a sample loading groove, a purification column and a collection area connected in sequence, and the collection area communicates with the reaction chamber.

[0032] Preferably, the sample loading hole is one of oval, circular, rectangular and square.

[0033] Furthermore, the purification column is one of porous capillary channel type, gel filtration chromatography type, adsorption chromatography type, solid phase extraction type and magnetic bead purification type.

[0034] In a second aspect, a method for using the multi-color fluorescence liquid-phase detection system as described in the first aspect is provided, including the following steps:

[0035] S1. Add the sample to be detected from the sample loading hole to the sample loading groove;

[0036] S2. After the sample to be detected is purified by the purification column, it enters the reaction chamber through the collection area;

[0037] S3. After the sample to be detected specifically binds to the antibody carrying the marker in the reaction chamber, corresponding color signals are shown, and the presence or absence of the target is judged by observing the color on the side wall of the reaction chamber.

[0038] The beneficial effects of the present invention are as follows:

[0039] The present invention provides a multi-color fluorescence liquid-phase detection system and a method for using the same. After specific reactions occur between the antibodies carrying markers and the targets of the corresponding samples to be detected, different color signals are presented. By observing the color on the side wall of the reaction chamber, highly sensitive and multi-target intuitive visual detection can be realized. Utilizing the high-specific antibody immobilization ability of Protein A / G and combining with an efficient purification column, multiple different targets can be detected simultaneously, which has the advantages of simple operation and comprehensive detection, solves the limitations and preparation cost problems of traditional single-item detection systems, realizes multi-channel signal detection, and provides innovative technical support for precision medicine, individualized treatment and biomedical research. Description of the Drawings

[0040] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0041] Figure 1 It is a schematic structural diagram of the multi-color fluorescence liquid-phase detection system according to Embodiment 1 of the present invention;

[0042] Figure 2 It is a schematic cross-sectional structural diagram of the multi-color fluorescence liquid-phase detection system according to Embodiment 1 of the present invention;

[0043] Explanation of the markings in the figure:

[0044] 1 - Sampling hole; 2 - Sampling groove; 3 - Purification column; 4 - Collection area; 5 - Reaction chamber; 6 - First reaction surface; 7 - Second reaction surface; 8 - Third reaction surface; 9 - Fourth reaction surface. Specific embodiments

[0045] The following will clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0046] It should be understood that when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, wholes, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components, and / or their combinations.

[0047] It should also be understood that the terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. As used in this specification of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" are intended to include the plural forms.

[0048] It should be further understood that the term " / and / " used in this specification of the present invention and the appended claims refers to any combination and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0049] To more fully understand the technical content of the present invention, the following further introduces and illustrates the technical solutions of the present invention in combination with specific embodiments.

[0050] Embodiment 1

[0051] Please refer to Figure 1-2 the multi-color fluorescence liquid-phase detection system shown in the figure. The system includes a connected purification unit and a detection unit. The detection unit is arranged around the purification unit. The detection unit includes a plurality of reaction chambers 5. The side walls of each reaction chamber 5 are made of light-transmitting materials (aminated quartz glass) treated with Protein A. Antibodies carrying markers are coated on the side walls of the reaction chambers 5. Among them, the purification unit is used to remove impurities that interfere with the detection in the sample to be tested. The light-transmitting material can directly observe the color change of the detection result, facilitating the rapid reading of data and improving the convenience and visualization of detection.

[0052] The overall structure of the multi-color fluorescence liquid-phase detection system is a cuboid, and the purification unit is also a cuboid. The purification unit includes a sample loading hole 1, a sample loading groove 2, a purification column 3, and a collection area 4 connected in sequence from top to bottom. The collection area 4 is communicated with the reaction chamber 5. The sample loading hole 1 is circular. The purification column 3 is of a porous capillary channel type with a pore diameter of 0.22 - 0.45 μm.

[0053] In this embodiment, the reaction chamber 5 is composed of a first reaction chamber, a second reaction chamber, a third reaction chamber, and a fourth reaction chamber. The side wall of the first reaction chamber is the first reaction surface 6, the side wall of the second reaction chamber is the second reaction surface 7, the side wall of the third reaction chamber is the third reaction surface 8, and the side wall of the fourth reaction chamber is the fourth reaction surface 9. The marker is a latex microsphere, and the latex microsphere is specifically a polystyrene latex microsphere modified with amino groups. Among them, the first reaction surface 6 is coated with SARS-CoV-2 antibody carrying red latex microspheres as markers, the second reaction surface 7 is coated with Flu A antibody carrying yellow latex microspheres as markers, the third reaction surface 8 is coated with Flu B antibody carrying blue latex microspheres as markers, and the fourth reaction surface 9 is coated with RSV antibody carrying green latex microspheres as markers. Among them, the antibody carrying the marker is used to specifically bind to the target in the sample to be tested and then show the corresponding color signal. By using different antibodies carrying different color markers, the corresponding color signals are shown after specifically binding to the target in the sample to be tested, so as to achieve highly sensitive and multi-target visual detection. In this embodiment, the sample to be tested is an enterprise reference product (specifically diluted from virus cultures with negative nasal swab samples) respectively carrying SARS-CoV-2, Flu A, Flu B, and RSV.

[0054] The usage method of the multi-color fluorescence liquid-phase detection system includes the following steps:

[0055] S1. Add the sample to be tested from the sample loading hole 1 to the sample loading groove 2;

[0056] S2. After the sample to be tested is purified by the purification column 3, it enters the reaction chamber 5 through the collection area 4;

[0057] S3. After the sample to be tested specifically binds to the antibody carrying the marker in the reaction chamber 5, a corresponding color signal appears. The presence or absence of the target is judged by observing the color on the side wall of the reaction chamber 5. In this embodiment, when the sample to be tested is an enterprise reference product with SARS-CoV-2, the first reaction surface 6 of the first reaction chamber is red. When the sample to be tested is an enterprise reference product with Flu A, the second reaction surface 7 of the second reaction chamber is yellow. When the sample to be tested is an enterprise reference product with Flu B, the third reaction surface 8 of the third reaction chamber is blue. When the sample to be tested is an enterprise reference product with RSV, the fourth reaction surface 9 of the fourth reaction chamber is green.

[0058] Performance test

[0059] 1. Accuracy test

[0060] Two operators used the multi-color fluorescence liquid phase detection system to detect SARS-CoV-2, Flu A, Flu B, RSV reference products and negative reference products under 3 different environmental conditions (Environment 1: temperature is 4°C ± 1°C, humidity is 90% ± 5%; Environment 2: temperature is 25°C ± 1°C, humidity is 50% ± 5%; Environment 3: temperature is 45°C ± 1°C, humidity is 20% ± 5%), and each sample was repeated 3 times. The accuracy test results of the multi-color fluorescence liquid phase detection system in Example 1 are shown in Table 1.

[0061] Table 1 Accuracy test results of the multi-color fluorescence liquid phase detection system in Example 1

[0062]

[0063]

[0064] From the test results in Table 1, it can be seen that the sensitivity of the detection device is 95.8%, the specificity is 99.3%, the accuracy is 98.6%, and the Kappa value is 0.974.

[0065] 2. Minimum detection limit test

[0066] Three batches of multi-color fluorescence liquid phase detection systems were used to test SARS-CoV-2, Flu A, Flu B, and RSV samples at the minimum detection limit concentration. Each sample was repeatedly detected 20 times, and the positive detection rate should be ≥95%. The minimum detection limit test results of the multi-color fluorescence liquid phase detection system in Example 1 are shown in Table 2.

[0067] Table 2 Minimum detection limit test results of the multi-color fluorescence liquid phase detection system in Example 1

[0068]

[0069] As can be seen from the test results in Table 2, the multi-color fluorescence liquid-phase detection system of the embodiments of the present invention meets the detection requirements of the minimum detection limit.

[0070] 3. Repeatability test

[0071] Prepare high-negative (positive detection rate ≤ 5%), low-positive (positive detection rate ≥ 95%), and medium-positive (positive detection rate 100%) samples of SARS-CoV-2, Flu A, Flu B, and RSV. Two operators use three batches of multi-color fluorescence liquid-phase detection systems to continuously detect samples at three different concentration levels every day for five consecutive days in three different environments (Environment 1: temperature is 4°C ± 1°C, humidity is 90% ± 5%; Environment 2: temperature is 25°C ± 1°C, humidity is 50% ± 5%; Environment 3: temperature is 45°C ± 1°C, humidity is 20% ± 5%). Each batch of multi-color fluorescence liquid-phase detection systems is tested once, and each sample is repeated 90 times in total. Kappa should be not less than 0.75.

[0072] The results of the repeatability test (SARS-CoV-2) of the multi-color fluorescence liquid-phase detection system of Example 1 are shown in Table 3-1.

[0073] Table 3-1 Results of the repeatability test (SARS-CoV-2) of the multi-color fluorescence liquid-phase detection system of Example 1

[0074]

[0075]

[0076] The results of the repeatability test (Flu A) of the multi-color fluorescence liquid-phase detection system of Example 1 are shown in Table 3-2:

[0077] Table 3-2 Results of the repeatability test (Flu A) of the multi-color fluorescence liquid-phase detection system of Example 1

[0078]

[0079] The results of the repeatability test (Flu B) of the multi-color fluorescence liquid-phase detection system of Example 1 are shown in Table 3-3.

[0080] Table 3-3 Results of the repeatability test (Flu B) of the multi-color fluorescence liquid-phase detection system of Example 1

[0081]

[0082]

[0083] The results of the repeatability test (RSV) of the multi-color fluorescence liquid-phase detection system of Example 1 are shown in Table 3-4.

[0084] Table 3-4 Results of the repeatability test (RSV) of the multi-color fluorescence liquid-phase detection system of Example 1

[0085]

[0086]

[0087] From the test results in Table 3-1, Table 3-2, Table 3-3 and Table 3-4, it can be seen that the multi-color fluorescence liquid-phase detection system of the embodiment of the present invention meets the detection requirements of repeatability.

[0088] 4. Stability test

[0089] Prepare high-negative (positive detection rate ≤ 5%), low-positive (positive detection rate ≥ 95%) and medium-positive (positive detection rate 100%) samples of SARS-CoV-2, Flu A, Flu B, and RSV, and use three batches of the multi-color fluorescence liquid-phase detection system to repeat the detection 10 times.

[0090] The results of the stability test (SARS-CoV-2) of the multi-color fluorescence liquid-phase detection system of Example 1 are shown in Table 4-1.

[0091] Table 4-1 Results of the stability test (SARS-CoV-2) of the multi-color fluorescence liquid-phase detection system of Example 1

[0092]

[0093] The results of the stability test (Flu A) of the multi-color fluorescence liquid-phase detection system of Example 1 are shown in Table 4-2.

[0094] Table 4-2 Results of the stability test (Flu A) of the multi-color fluorescence liquid-phase detection system of Example 1

[0095]

[0096] The results of the stability test (Flu B) of the multi-color fluorescence liquid-phase detection system of Example 1 are shown in Table 4-3.

[0097] Table 4-3 Results of the stability test (Flu B) of the multi-color fluorescence liquid-phase detection system of Example 1

[0098]

[0099] The results of the stability test (RSV) of the multi-color fluorescence liquid-phase detection system of Example 1 are shown in Table 4-4.

[0100] Table 4-4 Results of the stability test (RSV) of the multi-color fluorescence liquid-phase detection system of Example 1

[0101]

[0102]

[0103] From the test results in Table 4-1, Table 4-2, Table 4-3 and Table 4-4, it can be seen that the multi-color fluorescence liquid-phase detection system of the embodiment of the present invention meets the detection requirements of stability.

[0104] 5. Reaction speed test

[0105] Start timing with a stopwatch (accuracy of 0.01 s) after adding the sample to be tested until the reaction surface shows color, and the time used is recorded as (t). Repeat the test 10 times and take the average value.

[0106] The test results of the reaction speed of the multi-color fluorescence liquid-phase detection system of Example 1 are shown in Table 5.

[0107] Table 5 Test results of the reaction speed of the multi-color fluorescence liquid-phase detection system of Example 1

[0108] test sample SARS-CoV-2 FluA FluB RSV 1 1294.94 1305.89 1256.43 1247.26 2 1383.93 1328.91 1290.05 1251.79 3 1341.32 1384.72 1242.95 1375.71 4 1290.34 1223.63 1369.09 1371.65 5 1229.85 1298.99 1347.66 1349.97 6 1223.09 1312.86 1201.33 1351.44 7 1244.81 1375.94 1224.78 1371.25 8 1380.35 1291.14 1341.81 1395.16 9 1202.16 1258.38 1333.91 1308.61 10 1327.40 1365.28 1368.87 1321.89 average value / s 1291.82 1314.57 1297.69 1334.47

[0109] From the test results in Table 5, it can be seen that the reaction speed of the multi-color fluorescence liquid-phase detection system of Example 1 is about 1300 s, and the detection time is shorter.

[0110] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of various equivalent modifications or substitutions, and these modifications or substitutions should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A multicolor fluorescent liquid phase detection system, characterized in that: It comprises a purification unit and a detection unit connected to each other, wherein the detection unit is arranged around the purification unit, and the detection unit comprises a plurality of reaction chambers, the side walls of each reaction chamber are made of a light-transmitting material treated with Protein A or Protein G, and the side walls of the reaction chambers are coated with antibodies carrying markers.

2. The multicolor fluorescent liquid phase detection system according to claim 1, characterized in that: The marker is one or more of fluorescent particles, enzymes, chemiluminescent substances, nanomaterials, magnetic particles, and rare earth ions.

3. The multicolor fluorescent liquid phase detection system according to claim 2, characterized in that: The antibodies are one or more of SARS-CoV-2 antibodies, Flu A antibodies, Flu B antibodies, and RSV antibodies.

4. The multicolor fluorescent liquid phase detection system according to claim 3, characterized in that: The light-transmitting material is one or more of glass, polycarbonate, polymethyl methacrylate, cycloolefin copolymer, and polydimethylsiloxane.

5. The multicolor fluorescent liquid phase detection system according to claim 4, characterized in that: The marker is a nano material, and the nano material is selected from one of carbon nano particles, non-metallic nano particles, nano metal particles, latex microspheres, and quantum dots.

6. The multicolor fluorescent liquid phase detection system according to claim 5, characterized in that: The antibody carrying the marker is used to specifically bind to the target in the sample to be tested and then display a corresponding color signal.

7. The multicolor fluorescent liquid phase detection system according to claim 6, characterized in that: The sample to be tested includes one or more of serum, plasma, whole blood, peripheral blood, urine, saliva, stool extract, oral cavity and human tissue extract.

8. The multicolor fluorescent liquid phase detection system according to claim 7, characterized in that: The purification unit comprises a sample loading hole, a sample loading groove, a purification column and a collection area which are connected in sequence, and the collection area is communicated with the reaction chamber.

9. The multicolor fluorescent liquid phase detection system according to claim 8, characterized in that: The purification column is one of a porous capillary channel type, a gel filtration chromatography type, an adsorption chromatography type, a solid phase extraction type, and a magnetic bead purification type.

10. The method for using the multicolor fluorescent liquid phase detection system according to any one of claims 7 to 9, characterized in that: The following steps are involved: S1. Add the sample to be tested from the sample adding hole to the sample adding slot; S2. After the sample is purified by the purification column, it enters the reaction chamber through the collection area; S3. After the sample to be tested specifically binds to the antibody carrying the marker in the reaction chamber, a corresponding color signal is displayed, and the presence or absence of the target is determined by observing the color of the side wall of the reaction chamber.

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