CN116879543A - Preparation method of GDF-15 detection card, kit and use method thereof - Google Patents

Preparation method of GDF-15 detection card, kit and use method thereof Download PDF

Info

Publication number
CN116879543A
CN116879543A CN202310842354.XA CN202310842354A CN116879543A CN 116879543 A CN116879543 A CN 116879543A CN 202310842354 A CN202310842354 A CN 202310842354A CN 116879543 A CN116879543 A CN 116879543A
Authority
CN
China
Prior art keywords
gdf
solution
detection
gold
monoclonal antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310842354.XA
Other languages
Chinese (zh)
Inventor
张云
张肖
吴晨
洪茂椿
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xiamen Institute of Rare Earth Materials
Original Assignee
Xiamen Institute of Rare Earth Materials
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Xiamen Institute of Rare Earth Materials filed Critical Xiamen Institute of Rare Earth Materials
Priority to CN202310842354.XA priority Critical patent/CN116879543A/en
Publication of CN116879543A publication Critical patent/CN116879543A/en
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Nanotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a preparation method of a GDF-15 detection card, a kit and a use method thereof, wherein a gold octahedral probe is used as a marker, the component of the gold octahedral probe is AuNO@4-MBA, wherein AuNO is gold octahedron, and@represents that a Raman signal molecule 4-mercaptobenzoic acid is modified on the surface of AuNO. The invention adopts the monoclonal antibody of the anti-GDF-15 marked by the gold octahedral probe marked by 4-MBA, the immune complex captures the other monoclonal antibody of the anti-GDF-15 fixed on the nitrocellulose membrane, the characteristic peak intensity of the detection line is read by a Raman spectrometer or the detection sensitivity of the sample is high according to the color development intensity, the negative positive can be directly judged according to the color development, and the invention has the characteristics of wide detection range, high sensitivity, high accuracy, rapid and simple detection and the like, and can be used for rapid detection.

Description

Preparation method of GDF-15 detection card, kit and use method thereof
Technical Field
The invention relates to a preparation method of a GDF-15 detection card, and belongs to the technical field of sample detection.
Background
Growth differentiation factor-15 (GDF-15) is an independent inflammation biochemical marker capable of reflecting cardiovascular functions and diseases, is closely related to the occurrence and development of Acute Coronary Syndrome (ACS), has great value in the aspect of risk stratification of ACS, and can provide decision support for long-term antithrombotic treatment of patients after ACS. GDF-15 has high sensitivity, can be detected in early myocardial injury, and can improve the diagnosis capability of ACS when being combined with traditional myocardial injury markers. The current detection technology of GDF-15 includes enzyme-linked immunosorbent assay, electrochemiluminescence method, fluorescence immunochromatography, colloidal gold immunochromatography and the like, wherein the enzyme-linked method and the electrochemiluminescence method need large-scale professional instruments; the colloidal gold chromatography is convenient and quick, can self-test and read, but has low sensitivity; fluorescence immunochromatography has high sensitivity and stability, but requires a matched instrument and cannot be visualized. The 202111345701.5 patent solves the problems of low detection range and insufficient sensitivity, but still requires the use of fluorescent equipment for the cooperation treatment, and still has a detection threshold. Therefore, the development of the chromatographic kit and the method which have high sensitivity, rapid determination, high detection accuracy of GDF-15 in serum, plasma and whole blood samples and can be read by naked eyes has important significance.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of a GDF-15 detection card, which comprises the following steps:
s1: preparing a bonding pad: adding a gold octahedral probe into the GDF-15 monoclonal antibody 1, blocking with BSA, centrifuging at a high speed, washing with a preservation solution, re-suspending, spraying a microsphere line on a binding pad by using a GDF-15 monoclonal antibody 1 solution marked by the gold octahedral probe, and drying overnight;
s2: preparing a coating film: respectively adopting GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody as detection lines and quality control lines to be drawn on the nitrocellulose membrane in parallel for coating, and drying overnight;
s3: preparing a sample pad: soaking the sample pad with the sample pad treatment liquid and drying the sample pad overnight;
s4: and sequentially and mutually bonding a sample pad, a bonding pad, a coating film and absorbent paper on the substrate in an overlapping manner to obtain a test paper board, and cutting to obtain the GDF-15 detection card.
Further, the gold octahedral probe in the step S1 has an AuNO@4-MBA, wherein AuNO is gold octahedron, and@represents that a Raman signal molecule 4-mercaptobenzoic acid is modified on the surface of AuNO.
Further, the GDF-15 monoclonal antibody 1 is added in the step S1 in an amount of 5 to 20. Mu.g/mL, and the BSA concentration is 20%.
Further, the sample pad treatment solution in the step S3 comprises 0.5% NaCl, 0.5% S17, 1% BSA, and 20mM Tris-HCl with pH of 8.0 of 1mg/ml anti-RBC antibody.
Further, the GDF-15 monoclonal antibody 2 coating concentration in the detection line is 0.1-2 mg/ml, the dosage is 0.5-1.5 mu l coating liquid amount/cm membrane, and the goat anti-mouse IgG antibody coating concentration in the quality control line is 0.5-2 mg/ml, the dosage is 0.5-1.5 mu l coating liquid amount/cm membrane.
Further, the GDF-15 monoclonal antibody 1 solution comprises buffer solution and GDF-15 monoclonal antibody, the microsphere diluent comprises 20-100 mM of 1% BSA and 2% sucrose, pH 7.5-8.5, buffer solution: GDF-15 monoclonal antibody = 1: (4-10).
Further, the invention also provides a preparation method of the Jin Bamian body probe, which comprises the following steps:
a1: synthesizing gold nanorods: adding chloroauric acid into cetyltrimethylammonium bromide (CTAB) under magnetic stirring, adding sodium borohydride solution for dissolution, and standing for 30min after stirring;
a2: preparing a growth solution: adding CTAB and sodium oleate into ultrapure water for dissolution, injecting chloroauric acid solution, adding silver nitrate solution after colorless solution, stirring, adding hydrochloric acid and ascorbic acid, adding seed solution after reaction, standing the reaction mixture for 12 hours, and performing centrifugal cleaning and then dispersing in ultrapure water;
A3: synthesis of gold nano octahedron: mixing CTAB and chloroauric acid, heating in a water bath at 60 ℃, adding 3-butenoic acid, adding gold nanorods after the reaction is colorless, and standing for 12 hours in the water bath at 60 ℃ to obtain a gold octahedral solution;
a4: modification of raman signal molecules: adding 4-mercaptobenzoic acid (4-MBA) solution into the gold octahedral solution, standing for 4 hours, centrifuging to remove redundant 4-MBA, and re-suspending in pure water.
The invention also provides a GDF-15 detection kit, and the detection card is prepared by adopting the preparation method of the GDF-15 detection card.
The using method of the GDF-15 detection kit comprises the following steps: taking 80 mu L of a sample, placing the sample on a sample pad, visually judging the sample after 15 minutes, and using a portable Raman spectrometer of 785nm laser to collect Raman signals of a detection line of a test strip by using detection parameters of 2 times of accumulation with 8.4mW laser power and 5s integration time, and testing; in the test process, the BWSpec4.0 software is used for carrying out the base line and smooth noise reduction treatment on the original data, and the origin software is used for carrying out curve fitting on the treated data.
By adopting the technical scheme, the invention has the following beneficial effects:
(1) The immune complex is captured by another anti-GDF-15 monoclonal antibody fixed on a nitrocellulose membrane by adopting a 4-MBA-marked gold octahedral probe-marked anti-GDF-15 monoclonal antibody, the characteristic peak intensity of a detection line is read by means of a Raman spectrometer or the detection sensitivity of a sample is high according to the color development intensity, negative positive can be directly judged according to the color development, and the immune complex has the characteristics of wide detection range, high sensitivity, high accuracy, rapid and simple detection and the like, and can be used for rapid detection.
(2) The gold nanorod which is easy to prepare is used as a core, and grows into the gold nano octahedron, so that the metal substrate with extremely strong Raman enhancement effect can be simply prepared, the manufacturing process is more stable and convenient, and the industrialization of the high-sensitivity Raman test strip is possible.
(3) The nano probe adopted by the invention is a gold octahedral probe and has Surface Enhanced Raman Scattering (SERS). SERS has unique bioanalytical advantages over traditional bioanalytical methods, including: high sensitivity; compared with fluorescence, the fluorescent dye has the capabilities of resisting photobleaching and photodegradation, and is suitable for long-term monitoring; the bandwidth of the spectrum is narrow, and the raman characteristic peak is usually very narrow, 10-100 times narrower than the fluorescence emission peak of the organic dye or quantum dot.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which
FIG. 1 is a view of a gold octahedral probe transmission electron microscope;
FIG. 2 is a schematic diagram of the structure of a GDF-15 cartridge of the present invention;
FIG. 3 is a standard curve of the average value of the Raman characteristic peaks;
fig. 4 is a linear analysis of the concentration detection results.
Fig. 5 is a photograph of the test paper visually observed.
The reference numerals are: the device comprises a substrate 1, a coating film 2, a detection line 21, a quality control line 22, a bonding pad 3, a sample pad 4 and absorbent paper 5.
Detailed Description
In order to better understand the above technical solutions, the following detailed description will refer to the accompanying drawings and specific embodiments.
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures.
In the description of the embodiments of the present invention, it should be understood that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," "outer," and the like indicate orientations or positional relationships based on those shown in the drawings, or those conventionally put in place when the inventive product is used, or those conventionally understood by those skilled in the art, merely for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the apparatus or element to be referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention.
In the description of the embodiments of the present invention, it should also be noted that, unless explicitly specified and limited otherwise, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art. The invention is further described below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
Example 1
The preparation method of the GDF-15 detection card comprises the following steps:
s1: preparing a bonding pad 3: adding GDF-15 monoclonal antibody 1 into gold octahedral probe according to 20 mug/mL, mixing uniformly, standing for reaction for 10min, adding 10 mug/mL of 20% BSA for blocking, mixing uniformly, standing for reaction for 5min, centrifuging at 10000rpm for 10min, washing with 20mM containing 1% BSA and PBS buffer solution with pH of 8.0, re-suspending to 20% of the original volume, diluting the GDF-15 monoclonal antibody 1 marked with gold octahedral probe on a binding pad with 20mM containing 0.5% Tween-20,2% BSA and 5% sucrose, diluting with microsphere with PBS buffer solution with pH of 8.0 for 4 times, uniformly spraying a line with the dosage of 2 mug of liquid volume/cm, placing in an oven, drying overnight at 37 ℃ and drying overnight at 37 ℃;
s2: preparing a coating film 2: respectively using GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody as detection line quality control lines to be drawn on a nitrocellulose membrane in parallel for coating, and drying; GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody are respectively regulated to 0.5mg/ml by using 10mM PB coating liquid containing 2.5% trehalose, the coating liquid amount/cm film is 0.5 mu l, the film is respectively used as a detection line and a quality control line to be parallelly marked on a nitrocellulose film for coating, the space between the quality control line and the detection line is 3mM, and the film is placed in an oven for drying overnight at 45 ℃;
S3: sample pad 4 was prepared: uniformly spraying two lines on the side, close to the binding pad, of a sample pad treatment solution containing 20mM of anti-RBC antibody and Tris-HCl with pH of 8.0, wherein the side is close to the binding pad, of the sample pad treatment solution containing 0.5% of NaCl, 0.5% of S17, 1% of BSA and 1mg/ml of anti-RBC antibody in parallel, and drying at 37 ℃ overnight;
s4: sample pads 4 (size 23 x 300mm, glass wool material), bonding pads 3 (size 10 x 300mm, glass wool material), coating film 2 (size 25 x 300mm, nitrocellulose material) and absorbent paper 5 (size 28 x 300 mm) were sequentially adhered to a base sheet 1 (size 80 x 300 mm) in a lap joint manner to obtain test paper boards, and the test paper boards were cut into GDF-15 test cards having a width of 4mm as required.
The content of the GDF-15 monoclonal antibody 1 marked by the gold octahedral probe sprayed on the bonding pad 2 is 5-20 mug antibody/mL gold octahedral probe.
The detection line is coated with GDF-15 monoclonal antibody 2 (1 mg/ml), and the quality control line is coated with goat anti-mouse IgG antibody with the concentration of 1mg/ml (wherein GDF-15 monoclonal antibody is purchased from Nanjing Bai Biotechnology Co., ltd., from Changshabo Biotechnology Co.). The amount of the microsphere was 4. Mu.l of coating solution/cm of sample pad, the amount of the detection line and the amount of the quality control line were 1. Mu.l of coating solution/cm of membrane.
The preparation method of the Jin Bamian body probe comprises the following steps:
a1: synthesizing gold nanorods: 250. Mu.L of 10mM chloroauric acid was added to 9.75mL of 0.1M cetyltrimethylammonium bromide (CTAB) with magnetic stirring in a vessel, followed by 0.6mL of 10mM sodium borohydride solution, stirred for 2min and then left at 28℃for 30min;
a2: growth liquid: 7.2g of CTAB and 0.987g of sodium oleate are added into 400mL of ultrapure water for dissolution, then 20mL of 10mM chloroauric acid is injected into the solution, 8mL of 10mM silver nitrate solution is added when the solution is colorless, 1.2mL of hydrochloric acid and 600 mu L of 0.1M ascorbic acid are added after stirring for 5min, 1mL of seed solution is added after reaction for about 30s, and the reaction mixture is left to stand at 30 ℃ for 12 hours to complete the synthesis of gold nanorods. After synthesis, the mixture is centrifuged at 9000rpm for 15min, and after centrifugal cleaning for 2 times, the mixture is dispersed in 20mL of ultrapure water;
a3: synthesis of gold nano octahedron: 50mL of 10mM CTAB and 500. Mu.L of 0.05M chloroauric acid are mixed, heated for 2min at 60 ℃ in a water bath, then 220. Mu.L of 3-butenoic acid is added into the mixture, 300. Mu.L of gold nanorods are added after the solution becomes colorless, and the mixture is kept stand for 12 hours at 60 ℃ in the water bath;
a4: modification of raman signal molecules: 5mL of gold octahedral solution was taken, and 250. Mu.L of 10 was added to the solution -4 The solution of M in 4-mercaptobenzoic acid (4-MBA) was left to stand for 4 hours and centrifuged to remove excess 4-MBA, which was then resuspended in purified water. And finishing modification of the Raman signal molecule, namely AuNO@4-MBA.
The GDF-15 detection kit is characterized in that the detection card is prepared by adopting the preparation method of the GDF-15 detection card.
The using method of the GDF-15 detection kit is characterized in that: taking 80 mu L of a sample, placing the sample on a sample pad, visually judging the sample after 15 minutes, and using a portable Raman spectrometer of 785nm laser to collect Raman signals of a detection line of a test strip by using detection parameters of 2 times of accumulation with 8.4mW laser power and 5s integration time, and testing; in the test process, the BWSpec4.0 software is used for carrying out the base line and smooth noise reduction treatment on the original data, and the origin software is used for carrying out curve fitting on the treated data.
Example 2
The preparation method of the GDF-15 detection card comprises the following steps:
s1: preparing a bonding pad 3: adding GDF-15 monoclonal antibody 1 into gold octahedral probe according to 20 mug/mL, mixing uniformly, standing for reaction for 10min, adding 10 mug/mL of 20% BSA for blocking, mixing uniformly, standing for reaction for 5min, centrifuging at 10000rpm for 10min, washing with 20mM containing 1% BSA and PBS buffer solution with pH of 8.0, re-suspending to 20% of the original volume, diluting the GDF-15 monoclonal antibody 1 marked with gold octahedral probe on a binding pad with 20mM containing 0.5% Tween-20,2% BSA and 5% sucrose, and microsphere dilution with PBS buffer solution with pH of 8.0 for 10 times, uniformly spraying a line with the dosage of 4 mug of liquid volume/cm, placing in an oven, drying overnight at 37 ℃ and drying overnight at 37 ℃;
S2: preparing a coating film 2: respectively using GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody as detection line quality control lines to be drawn on a nitrocellulose membrane in parallel for coating, and drying; GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody are respectively regulated to 2mg/ml by 10mM PB coating liquid containing 2.5% trehalose, the coating liquid amount/cm membrane is 1.5 mu l, the coating liquid amount/cm membrane is respectively used as a detection line and a quality control line to be parallelly marked on a nitrocellulose membrane for coating, the space between the quality control line and the detection line is 7mM, and the nitrocellulose membrane is placed in an oven for drying at 45 ℃ overnight;
s3: sample pad 4 was prepared: uniformly spraying two lines on the side, close to the binding pad, of a sample pad treatment solution containing 20mM of anti-RBC antibody and Tris-HCl with pH of 8.0, wherein the side is close to the binding pad, of the sample pad treatment solution containing 0.5% of NaCl, 0.5% of S17, 1% of BSA and 1mg/ml of anti-RBC antibody in parallel, and drying at 37 ℃ overnight;
s4: sample pads 4 (size 23 x 300mm, glass wool material), bonding pads 3 (size 10 x 300mm, glass wool material), coating film 2 (size 25 x 300mm, nitrocellulose material) and absorbent paper 5 (size 28 x 300 mm) were sequentially adhered to a base sheet 1 (size 80 x 300 mm) in a lap joint manner to obtain test paper boards, and the test paper boards were cut into GDF-15 test cards having a width of 4mm as required.
The content of the GDF-15 monoclonal antibody 1 marked by the gold octahedral probe sprayed on the binding pad 2 is 20 mug antibody/mL gold octahedral probe.
The detection line is coated with GDF-15 monoclonal antibody 2 (2 mg/ml), and the quality control line is coated with goat anti-mouse IgG antibody with the concentration of 2mg/ml (wherein GDF-15 monoclonal antibody is purchased from Nanjing Bai Biotechnology Co., ltd., from Changshabo Biotechnology Co.). The amount of the microsphere was 4. Mu.l of coating solution/cm of sample pad, the amount of the detection line and the amount of the quality control line were 1.5. Mu.l of coating solution/cm of membrane.
The preparation method of the Jin Bamian body probe comprises the following steps:
a1: synthesizing gold nanorods: 250. Mu.L of 10mM chloroauric acid was added to 9.75mL of 0.1M cetyltrimethylammonium bromide (CTAB) with magnetic stirring in a vessel, followed by 0.6mL of 10mM sodium borohydride solution, stirred for 2min and then left at 28℃for 30min;
a2: growth liquid: 7.2g of CTAB and 0.987g of sodium oleate are added into 400mL of ultrapure water for dissolution, then 20mL of 10mM chloroauric acid is injected into the solution, 8mL of 10mM silver nitrate solution is added when the solution is colorless, 1.2mL of hydrochloric acid and 600 mu L of 0.1M ascorbic acid are added after stirring for 5min, 1mL of seed solution is added after reaction for about 30s, and the reaction mixture is left to stand at 30 ℃ for 12 hours to complete the synthesis of gold nanorods. After synthesis, the mixture is centrifuged at 9000rpm for 15min, and after centrifugal cleaning for 2 times, the mixture is dispersed in 20mL of ultrapure water;
A3: synthesis of gold nano octahedron: 50mL of 10mM CTAB and 500. Mu.L of 0.05M chloroauric acid are mixed, heated for 2min at 60 ℃ in a water bath, then 220. Mu.L of 3-butenoic acid is added into the mixture, 300. Mu.L of gold nanorods are added after the solution becomes colorless, and the mixture is kept stand for 12 hours at 60 ℃ in the water bath;
a4: modification of raman signal molecules: 5mL of gold octahedral solution was taken, and 250. Mu.L of 10 was added to the solution -4 The solution of M in 4-mercaptobenzoic acid (4-MBA) was left to stand for 4 hours and centrifuged to remove excess 4-MBA, which was then resuspended in purified water. And finishing modification of the Raman signal molecule, namely AuNO@4-MBA.
The GDF-15 detection kit is characterized in that the detection card is prepared by adopting the preparation method of the GDF-15 detection card.
The using method of the GDF-15 detection kit is characterized in that: taking 80 mu L of a sample, placing the sample on a sample pad, visually judging the sample after 15 minutes, and using a portable Raman spectrometer of 785nm laser to collect Raman signals of a detection line of a test strip by using detection parameters of 2 times of accumulation with 8.4mW laser power and 5s integration time, and testing; in the test process, the BWSpec4.0 software is used for carrying out the base line and smooth noise reduction treatment on the original data, and the origin software is used for carrying out curve fitting on the treated data.
Example 3
The preparation method of the GDF-15 detection card comprises the following steps:
s1: preparing a bonding pad 3: adding GDF-15 monoclonal antibody 1 into gold octahedral probe according to 20 mug/mL, mixing uniformly, standing for reaction for 10min, adding 10 mug/mL of 20% BSA for blocking, mixing uniformly, standing for reaction for 5min, centrifuging at 10000rpm for 10min, washing with 20mM containing 1% BSA and PBS buffer solution with pH of 8.0, re-suspending to 20% of the original volume, diluting the GDF-15 monoclonal antibody 1 marked with gold octahedral probe on a binding pad with 20mM containing 0.5% Tween-20,2% BSA and 5% sucrose, and microsphere dilution with PBS buffer solution with pH of 8.0 for 4 times, uniformly spraying a line with the dosage of 4 mug of liquid volume/cm, placing in an oven, drying overnight at 37 ℃ and drying overnight at 37 ℃;
s2: preparing a coating film 2: respectively using GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody as detection line quality control lines to be drawn on a nitrocellulose membrane in parallel for coating, and drying; GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody are respectively regulated to 0.5mg/ml by using 10mM PB coating liquid containing 2.5% trehalose, the coating liquid amount/cm membrane is 1.5 mu l, the coating liquid amount/cm membrane is respectively used as a detection line and a quality control line to be parallelly marked on a nitrocellulose membrane for coating, the space between the quality control line and the detection line is 3mM, and the nitrocellulose membrane is placed in an oven for drying at 45 ℃ overnight;
S3: sample pad 4 was prepared: uniformly spraying two lines on the side, close to the binding pad, of a sample pad treatment solution containing 20mM of anti-RBC antibody and Tris-HCl with pH of 8.0, wherein the side is close to the binding pad, of the sample pad treatment solution containing 0.5% of NaCl, 0.5% of S17, 1% of BSA and 1mg/ml of anti-RBC antibody in parallel, and drying at 37 ℃ overnight;
s4: sample pads 4 (size 23 x 300mm, glass wool material), bonding pads 3 (size 10 x 300mm, glass wool material), coating film 2 (size 25 x 300mm, nitrocellulose material) and absorbent paper 5 (size 28 x 300 mm) were sequentially adhered to a base sheet 1 (size 80 x 300 mm) in a lap joint manner to obtain test paper boards, and the test paper boards were cut into GDF-15 test cards having a width of 4mm as required.
Wherein, the sample pad 4 is sprayed with microsphere lines, and the Jin Bamian body probe marked GDF-15 monoclonal antibody 1; the coating film 2 is provided with a detection line 21 and a quality control line 22, the detection line 21 is close to the bonding pad 3, and the detection line 21 and the quality control line 22 are parallel to each other and are spaced at a distance of 5mm; the detection line 21 is coated with GDF-15 monoclonal antibody 2, and the quality control line 22 is coated with goat anti-mouse IgG antibody.
The content of the GDF-15 monoclonal antibody 1 marked by the gold octahedral probe sprayed on the binding pad 2 is 5 mug antibody/mL gold octahedral probe.
The detection line is coated with GDF-15 monoclonal antibody 2 (0.5 mg/ml), and the quality control line is coated with goat anti-mouse IgG antibody with the concentration of 0.5mg/ml (wherein GDF-15 monoclonal antibody is purchased from Nanjing Bai Biotechnology Co., ltd., from Changshabo Biotechnology Co.). The amount of the microsphere was 4. Mu.l of coating solution/cm of sample pad, the amount of the detection line and the amount of the quality control line were 1.5. Mu.l of coating solution/cm of membrane.
The preparation method of the Jin Bamian body probe comprises the following steps:
a1: synthesizing gold nanorods: 250. Mu.L of 10mM chloroauric acid was added to 9.75mL of 0.1M cetyltrimethylammonium bromide (CTAB) with magnetic stirring in a vessel, followed by 0.6mL of 10mM sodium borohydride solution, stirred for 2min and then left at 28℃for 30min;
a2: growth liquid: 7.2g of CTAB and 0.987g of sodium oleate are added into 400mL of ultrapure water for dissolution, then 20mL of 10mM chloroauric acid is injected into the solution, 8mL of 10mM silver nitrate solution is added when the solution is colorless, 1.2mL of hydrochloric acid and 600 mu L of 0.1M ascorbic acid are added after stirring for 5min, 1mL of seed solution is added after reaction for about 30s, and the reaction mixture is left to stand at 30 ℃ for 12 hours to complete the synthesis of gold nanorods. After synthesis, the mixture is centrifuged at 9000rpm for 15min, and after centrifugal cleaning for 2 times, the mixture is dispersed in 20mL of ultrapure water;
A3: synthesis of gold nano octahedron: 50mL of 10mM CTAB and 500. Mu.L of 0.05M chloroauric acid are mixed, heated for 2min at 60 ℃ in a water bath, then 220. Mu.L of 3-butenoic acid is added into the mixture, 300. Mu.L of gold nanorods are added after the solution becomes colorless, and the mixture is kept stand for 12 hours at 60 ℃ in the water bath;
a4: modification of raman signal molecules: 5mL of gold octahedral solution was taken, and 250. Mu.L of 10 was added to the solution -4 The solution of M in 4-mercaptobenzoic acid (4-MBA) was left to stand for 4 hours and centrifuged to remove excess 4-MBA, which was then resuspended in purified water. And finishing modification of the Raman signal molecule, namely AuNO@4-MBA.
The GDF-15 detection kit is characterized in that the detection card is prepared by adopting the preparation method of the GDF-15 detection card.
The using method of the GDF-15 detection kit is characterized in that: taking 80 mu L of a sample, placing the sample on a sample pad, visually judging the sample after 15 minutes, and using a portable Raman spectrometer of 785nm laser to collect Raman signals of a detection line of a test strip by using detection parameters of 2 times of accumulation with 8.4mW laser power and 5s integration time, and testing; in the test process, the BWSpec4.0 software is used for carrying out the base line and smooth noise reduction treatment on the original data, and the origin software is used for carrying out curve fitting on the treated data.
Example 4
The preparation method of the GDF-15 detection card comprises the following steps:
s1: preparing a bonding pad 3: adding GDF-15 monoclonal antibody 1 into gold octahedral probe according to 20 mug/mL, mixing uniformly, standing for reaction for 10min, adding 10 mug/mL of 20% BSA for blocking, mixing uniformly, standing for reaction for 5min, centrifuging at 10000rpm for 10min, washing with 20mM containing 1% BSA and PBS buffer solution with pH of 8.0, re-suspending to 20% of the original volume, diluting the GDF-15 monoclonal antibody 1 marked with gold octahedral probe on a binding pad with 20mM containing 0.5% Tween-20,2% BSA and 5% sucrose, and microsphere dilution with PBS buffer solution with pH of 8.0 for 4 times, uniformly spraying a line with the dosage of 4 mug of liquid volume/cm, placing in an oven, drying overnight at 37 ℃ and drying overnight at 37 ℃;
s2: preparing a coating film 2: respectively using GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody as detection line quality control lines to be drawn on a nitrocellulose membrane in parallel for coating, and drying; GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody are respectively regulated to 0.5mg/ml by using 10mM PB coating liquid containing 2.5% trehalose, the coating liquid amount/cm film is 0.5 mu l, the film is respectively used as a detection line and a quality control line to be parallelly marked on a nitrocellulose film for coating, the space between the quality control line and the detection line is 7mM, and the film is placed in an oven for drying overnight at 45 ℃;
S3: sample pad 4 was prepared: uniformly spraying two lines on the side, close to the binding pad, of a sample pad treatment solution containing 20mM of anti-RBC antibody and Tris-HCl with pH of 8.0, wherein the side is close to the binding pad, of the sample pad treatment solution containing 0.5% of NaCl, 0.5% of S17, 1% of BSA and 1mg/ml of anti-RBC antibody in parallel, and drying at 37 ℃ overnight;
s4: sample pads 4 (size 23 x 300mm, glass wool material), bonding pads 3 (size 10 x 300mm, glass wool material), coating film 2 (size 25 x 300mm, nitrocellulose material) and absorbent paper 5 (size 28 x 300 mm) were sequentially adhered to a base sheet 1 (size 80 x 300 mm) in a lap joint manner to obtain test paper boards, and the test paper boards were cut into GDF-15 test cards having a width of 4mm as required.
Wherein, the sample pad 4 is sprayed with microsphere lines, and the Jin Bamian body probe marked GDF-15 monoclonal antibody 1; the coating film 2 is provided with a detection line 21 and a quality control line 22, the detection line 21 is close to the bonding pad 3, and the detection line 21 and the quality control line 22 are parallel to each other and are spaced at a distance of 7mm; the detection line 21 is coated with GDF-15 monoclonal antibody 2, and the quality control line 22 is coated with goat anti-mouse IgG antibody.
The content of the GDF-15 monoclonal antibody 1 marked by the gold octahedral probe sprayed on the binding pad 2 is 20 mug antibody/mL gold octahedral probe.
The detection line is coated with GDF-15 monoclonal antibody 2 (0.5 mg/ml), and the quality control line is coated with goat anti-mouse IgG antibody with the concentration of 0.5mg/ml (wherein GDF-15 monoclonal antibody is purchased from Nanjing Bai Biotechnology Co., ltd., from Changshabo Biotechnology Co.). The amount of the microsphere was 4. Mu.l of the coating liquid per cm of the sample pad, the amount of the detection line and the amount of the quality control line were 0.5. Mu.l of the coating liquid per cm of the membrane.
The preparation method of the Jin Bamian body probe comprises the following steps:
a1: synthesizing gold nanorods: 250. Mu.L of 10mM chloroauric acid was added to 9.75mL of 0.1M cetyltrimethylammonium bromide (CTAB) with magnetic stirring in a vessel, followed by 0.6mL of 10mM sodium borohydride solution, stirred for 2min and then left at 28℃for 30min;
a2: growth liquid: 7.2g of CTAB and 0.987g of sodium oleate are added into 400mL of ultrapure water for dissolution, then 20mL of 10mM chloroauric acid is injected into the solution, 8mL of 10mM silver nitrate solution is added when the solution is colorless, 1.2mL of hydrochloric acid and 600 mu L of 0.1M ascorbic acid are added after stirring for 5min, 1mL of seed solution is added after reaction for about 30s, and the reaction mixture is left to stand at 30 ℃ for 12 hours to complete the synthesis of gold nanorods. After synthesis, the mixture is centrifuged at 9000rpm for 15min, and after centrifugal cleaning for 2 times, the mixture is dispersed in 20mL of ultrapure water;
A3: synthesis of gold nano octahedron: 50mL of 10mM CTAB and 500. Mu.L of 0.05M chloroauric acid are mixed, heated for 2min at 60 ℃ in a water bath, then 220. Mu.L of 3-butenoic acid is added into the mixture, 300. Mu.L of gold nanorods are added after the solution becomes colorless, and the mixture is kept stand for 12 hours at 60 ℃ in the water bath;
a4: modification of raman signal molecules: 5mL of gold octahedral solution was taken, and 250. Mu.L of 10 was added to the solution -4 The solution of M in 4-mercaptobenzoic acid (4-MBA) was left to stand for 4 hours and centrifuged to remove excess 4-MBA, which was then resuspended in purified water. And finishing modification of the Raman signal molecule, namely AuNO@4-MBA.
The GDF-15 detection kit is characterized in that the detection card is prepared by adopting the preparation method of the GDF-15 detection card.
The using method of the GDF-15 detection kit is characterized in that: taking 80 mu L of a sample, placing the sample on a sample pad, visually judging the sample after 15 minutes, and using a portable Raman spectrometer of 785nm laser to collect Raman signals of a detection line of a test strip by using detection parameters of 2 times of accumulation with 8.4mW laser power and 5s integration time, and testing; in the test process, the BWSpec4.0 software is used for carrying out the base line and smooth noise reduction treatment on the original data, and the origin software is used for carrying out curve fitting on the treated data.
Example 5
The preparation method of the GDF-15 detection card comprises the following steps:
s1: preparing a bonding pad 3: GDF-15 monoclonal antibody 1 is added to the gold octahedral probe according to 20 mug/mL, the mixture is uniformly mixed, the mixture is kept stand and reacted for 10min, 10 mug/mL of 20% BSA is added for blocking, the mixture is uniformly mixed, the mixture is kept stand and reacted for 5min, the mixture is centrifuged at 10000rpm for 10min, the mixture is washed with 20mM containing 1% BSA and PBS buffer solution with pH of 8.0, and resuspended to 20% of the original volume, GDF-15 monoclonal antibody 1 marked with gold octahedral probe on the binding pad is diluted with 20mM containing 0.5% Tween-20,2% BSA and 5% sucrose, and microsphere dilution solution with PBS buffer solution with pH of 8.0 for 7 times, a line is uniformly sprayed, the amount of 2 mug of liquid is used for sample pad, the mixture is placed in an oven, and dried overnight at 37 ℃ for 37 degrees.
S2: preparing a coating film 2: respectively using GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody as detection line quality control lines to be drawn on a nitrocellulose membrane in parallel for coating, and drying; GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody are respectively regulated to 2mg/ml by 10mM PB coating liquid containing 2.5% trehalose, the coating liquid amount/cm membrane is 0.5 mu l, the coating liquid amount/cm membrane is respectively used as a detection line and a quality control line to be parallelly marked on a nitrocellulose membrane for coating, the space between the quality control line and the detection line is 5mM, and the nitrocellulose membrane is placed in an oven for drying at 45 ℃ overnight;
S3: sample pad 4 was prepared: two lines were sprayed in parallel and evenly on the side of the sample pad treatment solution containing 0.5% NaCl, 0.5% S17, 1% BSA, 1mg/ml anti-RBC antibody, 20mM Tris-HCl, pH8.0, near the conjugate pad, in an amount of 2. Mu.l liquid volume/cm, and dried at 37℃overnight;
s4: sample pads 4 (size 23 x 300mm, glass wool material), bonding pads 3 (size 10 x 300mm, glass wool material), coating film 2 (size 25 x 300mm, nitrocellulose material) and absorbent paper 5 (size 28 x 300 mm) were sequentially adhered to a base sheet 1 (size 80 x 300 mm) in a lap joint manner to obtain test paper boards, and the test paper boards were cut into GDF-15 test cards having a width of 4mm as required.
The content of the GDF-15 monoclonal antibody 1 marked by the gold octahedral probe sprayed on the bonding pad 2 is 5-20 mug antibody/mL gold octahedral probe.
The detection line is coated with GDF-15 monoclonal antibody 2 (2 mg/ml), and the quality control line is coated with goat anti-mouse IgG antibody with the concentration of 2mg/ml (wherein GDF-15 monoclonal antibody is purchased from Nanjing Bai Biotechnology Co., ltd., from Changshabo Biotechnology Co.). The amount of the microsphere was 2. Mu.l of coating solution/cm of sample pad, the amount of the detection line and the amount of the quality control line were 0.5. Mu.l of coating solution/cm of membrane.
The preparation method of the Jin Bamian body probe comprises the following steps:
a1: synthesizing gold nanorods: 250. Mu.L of 10mM chloroauric acid was added to 9.75mL of 0.1M cetyltrimethylammonium bromide (CTAB) with magnetic stirring in a vessel, followed by 0.6mL of 10mM sodium borohydride solution, stirred for 2min and then left at 28℃for 30min;
a2: growth liquid: 7.2g of CTAB and 0.987g of sodium oleate are added into 400mL of ultrapure water for dissolution, then 20mL of 10mM chloroauric acid is injected into the solution, 8mL of 10mM silver nitrate solution is added when the solution is colorless, 1.2mL of hydrochloric acid and 600 mu L of 0.1M ascorbic acid are added after stirring for 5min, 1mL of seed solution is added after reaction for about 30s, and the reaction mixture is left to stand at 30 ℃ for 12 hours to complete the synthesis of gold nanorods. After synthesis, the mixture is centrifuged at 9000rpm for 15min, and after centrifugal cleaning for 2 times, the mixture is dispersed in 20mL of ultrapure water;
a3: synthesis of gold nano octahedron: 50mL of 10mM CTAB and 500. Mu.L of 0.05M chloroauric acid are mixed, heated for 2min at 60 ℃ in a water bath, then 220. Mu.L of 3-butenoic acid is added into the mixture, 300. Mu.L of gold nanorods are added after the solution becomes colorless, and the mixture is kept stand for 12 hours at 60 ℃ in the water bath;
a4: modification of raman signal molecules: 5mL of gold octahedral solution was taken, and 250. Mu.L of 10 was added to the solution -4 The solution of M in 4-mercaptobenzoic acid (4-MBA) was left to stand for 4 hours and centrifuged to remove excess 4-MBA, which was then resuspended in purified water. And finishing modification of the Raman signal molecule, namely AuNO@4-MBA.
The GDF-15 detection kit is characterized in that the detection card is prepared by adopting the preparation method of the GDF-15 detection card.
The using method of the GDF-15 detection kit is characterized in that: taking 80 mu L of a sample, placing the sample on a sample pad, visually judging the sample after 15 minutes, and using a portable Raman spectrometer of 785nm laser to collect Raman signals of a detection line of a test strip by using detection parameters of 2 times of accumulation with 8.4mW laser power and 5s integration time, and testing; in the test process, the BWSpec4.0 software is used for carrying out the base line and smooth noise reduction treatment on the original data, and the origin software is used for carrying out curve fitting on the treated data.
Example 6
The preparation method of the GDF-15 detection card comprises the following steps:
s1: preparing a bonding pad 3: GDF-15 monoclonal antibody 1 is added to the gold octahedral probe according to 20 mug/mL, the mixture is uniformly mixed, the mixture is kept stand and reacted for 10min, 10 mug/mL of 20% BSA is added for blocking, the mixture is uniformly mixed, the mixture is kept stand and reacted for 5min, the mixture is centrifuged at 10000rpm for 10min, the mixture is washed with 20mM containing 1% BSA and PBS buffer solution with pH of 8.0, and resuspended to 20% of the original volume, GDF-15 monoclonal antibody 1 marked with gold octahedral probe on the binding pad is diluted with 20mM containing 0.5% Tween-20,2% BSA and 5% sucrose, and microsphere dilution solution with PBS buffer solution with pH of 8.0 for 6 times, a line is uniformly sprayed, the amount of 4 mug of liquid is used for sample pad, the mixture is placed in an oven, and dried overnight at 37 ℃ for 37 degrees.
S2: preparing a coating film 2: respectively using GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody as detection line quality control lines to be drawn on a nitrocellulose membrane in parallel for coating, and drying; GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody are respectively regulated to 0.5mg/ml by using 10mM PB coating liquid containing 2.5% trehalose, the coating liquid amount/cm membrane is 1.5 mu l, the coating liquid amount/cm membrane is respectively used as a detection line and a quality control line to be parallelly marked on a nitrocellulose membrane for coating, the space between the quality control line and the detection line is 5mM, and the nitrocellulose membrane is placed in an oven for drying at 45 ℃ overnight;
s3: sample pad 4 was prepared: uniformly spraying two lines on the side, close to the binding pad, of a sample pad treatment solution containing 20mM of anti-RBC antibody and Tris-HCl with pH of 8.0, wherein the side is close to the binding pad, of the sample pad treatment solution containing 0.5% of NaCl, 0.5% of S17, 1% of BSA and 1mg/ml of anti-RBC antibody in parallel, and drying at 37 ℃ overnight;
s4: sample pads 4 (size 23 x 300mm, glass wool material), bonding pads 3 (size 10 x 300mm, glass wool material), coating film 2 (size 25 x 300mm, nitrocellulose material) and absorbent paper 5 (size 28 x 300 mm) were sequentially adhered to a base sheet 1 (size 80 x 300 mm) in a lap joint manner to obtain test paper boards, and the test paper boards were cut into GDF-15 test cards having a width of 4mm as required.
Wherein, the sample pad 4 is sprayed with microsphere lines, and the Jin Bamian body probe marked GDF-15 monoclonal antibody 1; the coating film 2 is provided with a detection line 21 and a quality control line 22, the detection line 21 is close to the bonding pad 3, and the detection line 21 and the quality control line 22 are parallel to each other and are spaced at a distance of 5mm; the detection line 21 is coated with GDF-15 monoclonal antibody 2, and the quality control line 22 is coated with goat anti-mouse IgG antibody.
The content of the GDF-15 monoclonal antibody 1 marked by the gold octahedral probe sprayed on the bonding pad 2 is 5-20 mug antibody/mL gold octahedral probe.
The detection line is coated with GDF-15 monoclonal antibody 2 (0.5 mg/ml), and the quality control line is coated with goat anti-mouse IgG antibody with the concentration of 0.5mg/ml (wherein GDF-15 monoclonal antibody is purchased from Nanjing Bai Biotechnology Co., ltd., from Changshabo Biotechnology Co.). The amount of the microsphere was 4. Mu.l of coating solution/cm of sample pad, the amount of the detection line and the amount of the quality control line were 1.5. Mu.l of coating solution/cm of membrane.
The preparation method of the Jin Bamian body probe comprises the following steps:
a1: synthesizing gold nanorods: 250. Mu.L of 10mM chloroauric acid was added to 9.75mL of 0.1M cetyltrimethylammonium bromide (CTAB) with magnetic stirring in a vessel, followed by 0.6mL of 10mM sodium borohydride solution, stirred for 2min and then left at 28℃for 30min;
A2: growth liquid: 7.2g of CTAB and 0.987g of sodium oleate are added into 400mL of ultrapure water for dissolution, then 20mL of 10mM chloroauric acid is injected into the solution, 8mL of 10mM silver nitrate solution is added when the solution is colorless, 1.2mL of hydrochloric acid and 600 mu L of 0.1M ascorbic acid are added after stirring for 5min, 1mL of seed solution is added after reaction for about 30s, and the reaction mixture is left to stand at 30 ℃ for 12 hours to complete the synthesis of gold nanorods. After synthesis, the mixture is centrifuged at 9000rpm for 15min, and after centrifugal cleaning for 2 times, the mixture is dispersed in 20mL of ultrapure water;
a3: synthesis of gold nano octahedron: 50mL of 10mM CTAB and 500. Mu.L of 0.05M chloroauric acid are mixed, heated for 2min at 60 ℃ in a water bath, then 220. Mu.L of 3-butenoic acid is added into the mixture, 300. Mu.L of gold nanorods are added after the solution becomes colorless, and the mixture is kept stand for 12 hours at 60 ℃ in the water bath;
a4: modification of raman signal molecules: 5mL of gold octahedral solution was taken, and 250. Mu.L of 10 was added to the solution -4 The solution of M in 4-mercaptobenzoic acid (4-MBA) was left to stand for 4 hours and centrifuged to remove excess 4-MBA, which was then resuspended in purified water. And finishing modification of the Raman signal molecule, namely AuNO@4-MBA.
The GDF-15 detection kit is characterized in that the detection card is prepared by adopting the preparation method of the GDF-15 detection card.
The using method of the GDF-15 detection kit is characterized in that: taking 80 mu L of a sample, placing the sample on a sample pad, visually judging the sample after 15 minutes, and using a portable Raman spectrometer of 785nm laser to collect Raman signals of a detection line of a test strip by using detection parameters of 2 times of accumulation with 8.4mW laser power and 5s integration time, and testing; in the test process, the BWSpec4.0 software is used for carrying out the base line and smooth noise reduction treatment on the original data, and the origin software is used for carrying out curve fitting on the treated data.
Wherein, SEM of the sample prepared in example 1 is shown in FIG. 1, and the structure of the prepared GDF-15 detection card is shown in FIG. 2.
Using the samples prepared in example 1, taking antigen calibrator and diluting with negative clinical samples respectively to obtain 30000, 20000, 12000, 6000, 1200, 600, 25pg/mL samples, repeatedly measuring three times each sample, taking T line Raman characteristic peak average value and calibrator concentration to establish a standard curve, and drawing the standard curve by using the antigen concentration and sample signal T line Raman characteristic peak average value, wherein curve data are shown in Table 1:
TABLE 1GDF-15 Standard Curve
The standard curve is shown in fig. 3. Wherein the GDF-15R values were 0.9985, respectively, and the concentration of GDF-15 contained in the sample was quantitatively determined by the reticle. The GDF-15 test card was tested for performance as follows:
(1) Minimum detection limit: repeated measurement is carried out for 20 times by using zero-value samples, the average value M and the standard deviation SD of the 20 times results are calculated, the blank average value is added with the detection limit (M+2SD) of the double standard deviation reporting method, the GDF-15 result is 21.54pg/mL, and the sensitivity standard is 25pg/mL.
(2) Linear range: and respectively taking 7 concentration values of GDF-1525-30000 pg/mL, repeatedly measuring each concentration for three times, and carrying out linear analysis on the average value of the measured concentrations in the theoretical concentration to obtain a GDF-15 linear equation y=0.9244x+382.29 and r=0.9970, which shows that the GDF-15 detection kit has good correlation in the linear range.
(3) Precision: three kits of this example were taken and assayed for CV in three batches of the repetitive property control, each kit was assayed 10 times in parallel with the repetitive property control, wherein GDF-15120000pg/mL of three batches had CV of 9.29%, 7.58% and 6.28% respectively, 1200pg/mL of three batches had CV of 7.16%, 6.22% and 7.95% respectively, and 9.77% of each batch had CV of 10% or less.
(4) Accuracy: selecting quality control substances of a basic sample as detection samples, dividing the detection samples into 3 parts with the same volume, respectively adding accuracy quality control substances with different concentrations into 2 parts of samples, preparing 2 recovery samples with different added concentrations, and calculating the concentration of the added object to be detected. The same amount of solution without the test object is added into another sample to prepare a basic sample. The sample was analyzed 3 times repeatedly and the mean value was calculated. Calculate recovery = (analysis sample concentration-base sample concentration)/addition concentration x 100%. The recovery rate of GDF-15 recovery sample 20000pg/mL was 96.22%, the recovery rate of 12000pg/mL was 91.49%, and the average recovery rate was 93.86%. The deviation is within 10%.
The sample of example 1 was continuously observed, and the change in color development on the T line was shown in fig. 5. The concentration from left to right was 30000, 20000, 12000, 6000, 1200, 600, 25,0pg/mL.
100 GDF-15 blood samples were collected from hospitals, and the kit of examples 1 to 6 of the present invention was used for detection and comparison with the kit for detecting growth differentiation factor-15 by direct chemiluminescence method of Mike organism Co., ltd. In the kit, 80 μl of a blood sample is taken and added into a sample adding hole of a detection card, the concentration is read by a Raman spectrometer after chromatography for 10min, and the same sample is subjected to concentration detection by adopting a growth differentiation factor-15 detection kit (direct chemiluminescence method) of a direct chemiluminescence method of a comparative system Mike organism Co. As shown in FIG. 4, the correlation of the detection results is good, GDF-15r=0.9850, and P >0.05, the average relative deviation is less than 10%, and the results meet the clinical analysis requirements, thus being suitable for clinical detection.
Therefore, the kit disclosed by the invention utilizes the sensitivity of the rare earth nano fluorescent immunochromatography technology and combines a dry immunofluorescence analyzer to realize high-sensitivity, accurate and quantitative, simple, convenient and rapid quantitative detection of the GDF-15 antibody.
While the foregoing is directed to embodiments of the present invention, other and further details of the invention may be had by the present invention, it should be understood that the foregoing description is merely illustrative of the present invention and that no limitations are intended to the scope of the invention, except insofar as modifications, equivalents, improvements or modifications are within the spirit and principles of the invention.

Claims (9)

1. A preparation method of a GDF-15 detection card is characterized in that: the method comprises the following steps:
s1: preparing a bonding pad: adding a gold octahedral probe into the GDF-15 monoclonal antibody 1, blocking with BSA, centrifuging at a high speed, washing with a preservation solution, re-suspending, spraying a microsphere line on a binding pad by using a GDF-15 monoclonal antibody 1 solution marked by the gold octahedral probe, and drying overnight;
s2: preparing a coating film: respectively adopting GDF-15 monoclonal antibody 2 and goat anti-mouse IgG antibody as detection lines and quality control lines to be drawn on the nitrocellulose membrane in parallel for coating, and drying overnight;
s3: preparing a sample pad: treating the sample pad with the sample pad treating liquid and drying overnight;
s4: and sequentially and mutually bonding a sample pad, a bonding pad, a coating film and absorbent paper on the substrate in an overlapping manner to obtain a test paper board, and cutting to obtain the GDF-15 detection card.
2. The method for preparing the GDF-15 cartridge as set forth in claim 1, wherein: the gold octahedral probe in the step S1 comprises the components of AuNO@4-MBA, wherein AuNO is gold octahedron, and@represents that a Raman signal molecule 4-mercaptobenzoic acid is modified on the surface of AuNO.
3. The method for preparing the GDF-15 cartridge as set forth in claim 1, wherein: the addition amount of the GDF-15 monoclonal antibody 1 in the step S1 is 5-20 mug/mL, and the BSA concentration is 20%.
4. The method for preparing the GDF-15 cartridge as set forth in claim 1, wherein: the sample pad treatment solution in the step S3 comprises 0.5% NaCl, 0.5% S17, 1% BSA, 1mg/ml 20mM Tris-HCl with pH of 8.0 of anti-RBC antibody.
5. The method for preparing the GDF-15 cartridge as set forth in claim 1, wherein: GDF-15 monoclonal antibody 2 coating concentration in the detection line is 0.1-2 mg/ml, the dosage is 0.5-1.5 mu l coating liquid amount/cm membrane, and sheep anti-mouse IgG antibody coating concentration in the quality control line is 0.5-2 mg/ml, the dosage is 0.5-1.5 mu l coating liquid amount/cm membrane.
6. The method for preparing the GDF-15 cartridge as set forth in claim 1, wherein: the GDF-15 monoclonal antibody 1 solution comprises buffer solution and GDF-15 monoclonal antibody, the microsphere diluent comprises 20-100 mM of 1% BSA and 2% sucrose, pH 7.5-8.5, buffer solution: GDF-15 monoclonal antibody = 1: (4-10).
7. The method for producing a GDF-15 cartridge according to any one of claims 1 to 6, wherein:
the preparation method of the Jin Bamian body probe comprises the following steps:
a1: synthesizing gold nanorods: adding chloroauric acid into cetyltrimethylammonium bromide (CTAB) under magnetic stirring, adding sodium borohydride solution for dissolution, and standing for 30min after stirring;
a2: preparing a growth solution: adding CTAB and sodium oleate into ultrapure water for dissolution, injecting chloroauric acid solution, adding silver nitrate solution after colorless solution, stirring, adding hydrochloric acid and ascorbic acid, adding seed solution after reaction, standing the reaction mixture for 12 hours, and performing centrifugal cleaning and then dispersing in ultrapure water;
a3: synthesis of gold nano octahedron: mixing CTAB and chloroauric acid, heating in a water bath at 60 ℃, adding 3-butenoic acid, adding gold nanorods after the reaction is colorless, and standing for 12 hours in the water bath at 60 ℃ to obtain a gold octahedral solution;
a4: modification of raman signal molecules: adding 4-mercaptobenzoic acid (4-MBA) solution into the gold octahedral solution, standing for 4 hours, centrifuging to remove redundant 4-MBA, and re-suspending in pure water.
8. A GDF-15 detection kit, wherein the detection card is prepared by the method for preparing the GDF-15 detection card according to any one of claims 1-6.
9. The method of claim 8, wherein the step of using the GDF-15 assay kit comprises: taking 80 mu L of a sample, placing the sample on a sample pad, visually judging the sample after 15 minutes, and using a portable Raman spectrometer of 785nm laser to collect Raman signals of a detection line of a test strip by using detection parameters of 2 times of accumulation with 8.4mW laser power and 5s integration time, and testing; in the test process, the BWSpec4.0 software is used for carrying out the base line and smooth noise reduction treatment on the original data, and the origin software is used for carrying out curve fitting on the treated data.
CN202310842354.XA 2023-07-11 2023-07-11 Preparation method of GDF-15 detection card, kit and use method thereof Pending CN116879543A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310842354.XA CN116879543A (en) 2023-07-11 2023-07-11 Preparation method of GDF-15 detection card, kit and use method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310842354.XA CN116879543A (en) 2023-07-11 2023-07-11 Preparation method of GDF-15 detection card, kit and use method thereof

Publications (1)

Publication Number Publication Date
CN116879543A true CN116879543A (en) 2023-10-13

Family

ID=88256158

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310842354.XA Pending CN116879543A (en) 2023-07-11 2023-07-11 Preparation method of GDF-15 detection card, kit and use method thereof

Country Status (1)

Country Link
CN (1) CN116879543A (en)

Similar Documents

Publication Publication Date Title
CN110376379B (en) Method for high-precision detection of CEA by combining molecular imprinting with silence area internal standard SERS technology
CN111334282B (en) PTH rare earth detection kit, PTH rare earth detection card, microsphere and preparation and detection methods thereof
CN108593920B (en) Immunosensor for detecting zearalenone and preparation method thereof
CN111208178B (en) Method for constructing electrochemical luminescence sensor based on double amplification of perylene tetracarboxylic acid signal by cobalt-based metal organic framework
CN108918853B (en) Pd @ Ag @ CeO2Preparation method and application of labeled immunosensor
Sun et al. A novel surface-enhanced Raman scattering method for simultaneous detection of ketamine and amphetamine
CN109444240B (en) Prussian blue-based electrochemical immunosensor, electrochemical immunosensing method established based on sensor and application
CN113433314B (en) Aflatoxin marker SERS side flow immunosensing analysis method based on visible nanogold detection line
CN116879543A (en) Preparation method of GDF-15 detection card, kit and use method thereof
CN110646616A (en) Hypersensitivity fluorescence quenching immunosensor for detecting human cTnI in serum and detection method
CN114994311A (en) SERS technology-based immune lateral flow test strip with self-calibration function and preparation method thereof
CN114965417A (en) Method for rapidly detecting methyl mercury by surface enhanced Raman scattering
CN113504377A (en) Test strip for bimodal detection of CRP (C-reactive protein), preparation method and detection method
Meng et al. Adriamycin coated silica microspheres as labels for cancer biomarker alpha-fetoprotein detection
CN114660293A (en) SERS immunoassay kit for detecting mycotoxin exposure marker in blood plasma or urine
Li et al. Two types of immunoassay based on Nile blue labeling polydopamine nanospheres
CN110470828B (en) Preparation method and detection method of cerebral apoplexy marker immunosensor
CN111504974A (en) Solution for vomitoxin detection, substrate preparation method and detection method
CN218496943U (en) Fluorescent immune test strip for detecting diarrhetic shellfish toxin
CN116930485B (en) Trace pollutant infrared signal enhancement and in-situ rapid detection method and detection system based on immune biological reaction
CN111678911B (en) SERS sensor based on capillary blotting and preparation method and application thereof
CN103529208A (en) Homogeneous-phase immunoassay method
CN116023693B (en) Carbon nano composite SERS substrate film and preparation method and application thereof
CN114217062B (en) Multicomponent homogeneous phase immunoassay method based on single particle inductively coupled plasma mass spectrometry
CN117330744A (en) Lateral flow immunochromatography test strip for detecting PSA (pressure sensitive adhesive) based on SERS (surface enhanced Raman scattering) technology and preparation and detection method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination