CN115575632A - Detection test strip and application - Google Patents
Detection test strip and application Download PDFInfo
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- CN115575632A CN115575632A CN202211560312.9A CN202211560312A CN115575632A CN 115575632 A CN115575632 A CN 115575632A CN 202211560312 A CN202211560312 A CN 202211560312A CN 115575632 A CN115575632 A CN 115575632A
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- 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
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- 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
- G01N33/54306—Solid-phase reaction mechanisms
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- 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
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- G—PHYSICS
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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Abstract
The invention belongs to the technical field of biological detection, and particularly discloses a detection test strip and application of the detection test strip in biological analysis and detection. In the detection test strip provided by the invention, the surface grafted linear polymer chain microspheres load capture antibody conjugates at the detection T line, and in the detection process, when a detection sample liquid flows through the detection T line, the surface grafted linear polymer chain microspheres extend to the periphery, and the loaded capture antibodies form a three-dimensional space antigen blocking net, so that the space contact efficiency of the antigen and the capture antibodies when the detection sample liquid flows through is remarkably improved, the detection sensitivity of the test strip is enhanced, and the detection is more accurate.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a detection test strip and application thereof in biological analysis and detection.
Background
The new coronavirus of the type has extremely high transmission rate, and needs to be quickly and sensitively detected at the source to inhibit the spread of epidemic situations. The traditional Polymerase Chain Reaction (PCR) is a gold standard for detecting the virus, the detection precision is high, and the sequence detection limit can reach 50 Copies/mL, but the method is based on Chain amplification detection, needs to be circularly heated and cooled, has a detection period as long as 60-90 minutes, and is not suitable for rapid detection of the virus. Therefore, for the virus cases with extremely high infection rate, a convenient, rapid and sensitive detection method is needed.
Antigen detection chromatography is a well-established commercial Point of Care Test (POCT) method, and has been widely used, for example, in the detection of human chorionic gonadotropin (pregnancy Test). The antigen detection chromatography is used for detecting virus antigens, and particularly has the following advantages for detecting respiratory system diseases with extremely strong infectivity such as novel coronavirus pneumonia: firstly, the antigen detection is to detect the Nucleocapsid protein (N protein) of the virus, and can detect the virus in the early stage of infection, so that the spread of the new coronavirus can be effectively controlled; secondly, the operation is convenient, the home self-test can be realized, the detection period is short, and only about 15 minutes are needed for detecting the sample; thirdly, the cost is low, and the cost of single detection is low. At present, the antigen chromatography test strip is widely applied to the detection of the novel coronavirus pneumonia.
However, although the antigen detection chromatography is a widely-proven rapid POCT detection method, it has the disadvantages of low sensitivity, easy occurrence of false negative phenomenon during detection, and inability of accurate detection, compared with the PCR detection technology. Therefore, for the detection of new coronavirus with extremely strong infectivity, the antigen detection chromatography still needs to further improve the sensitivity and specificity.
In order to improve the sensitivity of the antigen detection chromatography, many improvements have been made in terms of, for example, using a new detection material or changing the structure of the chromatographic strip. In the aspect of developing a new material for antigen detection, no report of successful application of the new material to antigen chromatography test strip detection is found. In terms of changing the structure of the chromatographic strip, for example, the detection is performed by using a "double chromatography", but additional equipment is required in the actual detection process, which causes inconvenience in the detection operation and increases the cost.
Therefore, the detection sensitivity is improved, accurate detection is realized, and the problem to be solved in the detection field of the current chromatography test strip is still solved urgently.
Disclosure of Invention
The invention mainly solves the technical problem of providing a detection test strip to improve the detection sensitivity and realize accurate detection.
In order to solve the above technical problems, in a first aspect, the present invention provides an application of a surface-grafted linear polymer chain microsphere in preparation of a test strip, preferably in preparation of a test line T of the test strip.
As a preferred embodiment of the present invention, the surface-grafted linear polymer chain microspheres are used as a coating material for a detection line T.
Further preferably, the surface-grafted linear polymer chain microspheres are used as a coating material for the detection line T after being bound with the capture antibody.
As a preferred embodiment of the present invention, the surface-grafted linear polymer chain microspheres comprise polymer microspheres and linear polymer chains grafted on the surfaces of the polymer microspheres.
As a further preferred embodiment of the present invention, the polymer microspheres are polystyrene microspheres, micro-nano carbon spheres or nano silica microspheres, and further preferred are polystyrene microspheres.
As a further preferred embodiment of the present invention, the linear polymer chain is a polymer having a reactive group and a charge, and is more preferably polyacrylic acid, polymethacrylic acid, polyacrylamide or polystyrene sulfonate, and is more preferably polyacrylic acid.
Preferably, the particle size of the surface grafting linear polymer chain microsphere is 100 to 400 nm.
As a preferred embodiment of the present invention, the surface-grafted linear polymer chain microspheres are bound to the capture antibody by electrostatic or chemical action.
In a second aspect, the invention provides a test strip, wherein a detection line T is disposed on the test strip, and the detection line T is coated with a capture antibody, and the capture antibody is bound on a surface-grafted linear polymer chain microsphere.
As a preferred embodiment of the invention, the detection line T is arranged on a nitrocellulose membrane, and a quality control line C is also arranged on the nitrocellulose membrane, and the quality control line C is coated with goat anti-mouse polyclonal IgG antibody.
Further preferably, the two ends of the nitrocellulose membrane are respectively provided with a gold combining pad and a water absorption pad, one end of the gold combining pad, which is far away from the nitrocellulose membrane, is provided with a sample pad, the quality control line C is close to the water absorption pad, and the detection line T and the quality control line C are arranged at intervals and in parallel.
As a specific embodiment of the invention, 1-2 mm are laminated between a sample pad and a gold seed combination pad, between the gold seed combination pad and a nitrocellulose membrane, and between the nitrocellulose membrane and a water absorption pad of the test strip, and the lengths of the sample pad, the gold seed combination pad, the nitrocellulose membrane and the water absorption pad are 10-17 mm.
The gold seed bonding pad is sprayed and marked with antibody nano colloidal gold for specifically recognizing the antigen to be detected, and preferably, the mouse anti-monoclonal detection antibody is marked with the nano colloidal gold.
The quality control line C is prepared by passing goat anti-mouse (multi-antibody) IgG antibody through a slide film of a scribing machine.
As an embodiment of the invention, the mouse anti-monoclonal antibody labeled nano colloidal gold is prepared by the following steps:
(1) Preparing colloidal gold nanoparticles AuNPs;
(2) The recognition antibody was labeled on the colloidal gold.
The colloidal gold nanoparticles AuNPs can be prepared by the following steps:
measuring ultrapure water by using an electronic balance, adding the ultrapure water into a round-bottom flask, adjusting an electric heating jacket to increase the temperature, and adding a chloroauric acid aqueous solution; and (3) after the solution in the round-bottom flask begins to boil, quickly adding the sodium citrate aqueous solution for one time, continuing to heat, stopping heating after the color turns to purple red, taking out the round-bottom flask, placing the round-bottom flask on a vortex instrument, and continuing to rotate until the solution is cooled, thus obtaining the colloidal gold nanoparticles AuNPs.
The method of labeling the recognition antibody on the colloidal gold may be an existing conventional method, for example: and (3) taking a colloidal gold solution containing colloidal gold nanoparticles AuNPs, uniformly mixing, adding a mouse anti-monoclonal detection antibody (D, namely the recognition antibody) for reaction, shaking uniformly for 5 minutes, and performing centrifugal separation to obtain the gold nanoparticles with the recognition antibodies marked on the surfaces.
The assembly process of the chromatographic detection test strip provided by the invention comprises the following steps: taking the nitrocellulose membrane as a boundary, sequentially attaching the pretreated gold seed combination pad and the sample pad to the left end, and attaching a water absorption pad to the right end of the nitrocellulose membrane; and the nitrocellulose membrane is sequentially scribed with a detection line T and a quality control line C.
The sample pad, the gold seed combination pad, the nitrocellulose membrane and the water absorption pad are all arranged on the viscous plastic substrate plate and sequentially comprise the sample pad, the gold seed combination pad, the nitrocellulose membrane and the water absorption pad from left to right.
In some embodiments, the length of the adhesive plastic substrate plate is 30 mm, the lengths of the sample pad, the gold bonding pad, the nitrocellulose membrane and the water absorption pad are between 10 and 17 mm, and the sample pad and the gold bonding pad, the gold bonding pad and the nitrocellulose membrane, and the nitrocellulose membrane and the water absorption pad are mutually laminated by 1-2 mm.
In a third aspect, the invention provides a method for preparing a test strip, comprising the steps of;
s1: preparing surface grafted linear polymer chain microspheres;
s2: preparing a conjugate of the surface grafted linear polymer chain microsphere and the capture antibody;
s3: and coating the conjugate of the surface grafted linear polymer chain microsphere and the capture antibody on a detection test strip to manufacture a detection line T.
Preferably, the preparation step of the surface-grafted linear polymer chain microspheres comprises:
s11, polymerizing styrene serving as a monomer in an inert atmosphere to obtain polystyrene microemulsion spheres;
s12, adding a photoinitiator into a polymerization reaction system at the final stage of the polymerization reaction in the step S11 to obtain spherical core emulsion coated by the photoinitiator;
s13, polymerizing the spherical core emulsion coated by the photoinitiator and an acrylic monomer, and performing polymerization reaction under ultraviolet irradiation in an inert atmosphere to obtain the surface grafted linear polymer chain microsphere;
further preferably, the using amount of the acrylic monomer is 35 to 80 percent of the solid content of the spherical core emulsion coated by the photoinitiator.
The photoinitiator is 2- (p-2-hydroxy-2-methyl propiophenone) -hydroxyethyl methacrylate (HMEM) or phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP).
Preferably, the preparation of the conjugate of the surface-grafted linear polymer chain microsphere and the capture antibody can be performed by electrostatic adsorption or chemical reaction.
Further preferably, the preparation of the conjugate by electrostatic interaction comprises the steps of:
mixing the surface-grafted linear polymer chain microspheres with excessive capture antibodies in a buffer solution, stirring, then adjusting the pH to the isoelectric point of the capture antibodies, spontaneously combining the surface-grafted linear polymer chain microspheres with the capture antibodies through electrostatic interaction, and fixing a large amount of capture antibodies in the surface-grafted linear polymer chain microspheres to form a soluble conjugate.
Further preferably, the preparation of the conjugate by chemical action comprises the steps of:
activating the surface grafted linear polymer chain microspheres by using a chemical cross-linking agent, then chemically combining with the capture antibody, and linking functional groups on the surface grafted linear polymer chain microspheres and amino residues of the capture antibody through chemical bonds to obtain a conjugate.
The chemical crosslinking agent is preferably 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), and more preferably the amount of the chemical crosslinking agent is equal to the number of carboxyl groups contained in the surface-grafted linear polymer chains in the surface-grafted linear polymer chain microspheres.
Preferably, the detection line T is prepared by coating the conjugate of the surface-grafted linear polymer chain microsphere and the capture antibody on a detection test strip, drying and curing.
Further preferably, the dosage of the surface grafting linear polymer chain microspheres used for coating the detection line T is 40-80 ug, and the concentration of the capture antibody is 400-1500 ug/mL.
In a fourth aspect, the invention provides a detection kit, which comprises the detection test strip of the invention.
In a fifth aspect, the invention provides a test strip or an application of the test kit in antigen or virus detection.
Preferably, the antigen is a recombinant N protein antigen.
Preferably, the virus is an inactivated strain, and further preferably is a wild strain of Alpha, beta, gamma, delta, BA1, BA2, WT.
In the detection test strip provided by the invention, the surface grafted linear polymer chain microspheres load capture antibody conjugates at the detection T line, and in the detection process, when a detection sample liquid flows through the detection T line, the surface grafted linear polymer chain microspheres extend to the periphery, and the loaded capture antibodies form a three-dimensional space antigen blocking net, so that the space contact efficiency of the antigen and the capture antibodies when the detection sample liquid flows through is remarkably improved, the detection sensitivity of the test strip is enhanced, and the detection is more accurate.
The surface of the surface-grafted linear polymer chain microsphere adopted in the invention is provided with a linear polymer chain with a large amount of charges, so that the surface-grafted linear polymer chain microsphere can be firmly combined with a capture antibody through electrostatic action or chemical action; and further coating the formed combination on a T line of a chromatographic test strip, wherein polymer chains with charges at the end parts of the surface grafted linear polymer chain microspheres are densely arranged on the surface of the spherical substrate, and due to the volume repulsion effect and the electrostatic repulsion effect, the polymer chains and chains, and the polymer beads can extend to the surrounding space, so that a support is formed. Compared with the traditional test strip which directly coats the capture antibody on the T line, when the test sample liquid flows to the T line, the polymer chains and the polymer beads can fully extend to the surrounding space, and a large amount of capture antibodies loaded on the test strip can form a three-dimensional space network structure, so that the space capture efficiency of the capture antibodies on the antigen at the T line is remarkably improved, and the detection sensitivity of the test strip is improved.
When the detection test strip provided by the invention is used for detection, the recombinant N protein antigen mother solution is diluted; then dripping the sample on a sample pad, allowing the sample to flow through a gold combining pad in the process of driving sample liquid chromatography by a water absorption pad, capturing the antigen to be detected by the colloidal gold surface recognition antibody, carrying out liquid chromatography to a detection T line, allowing the surface grafted linear polymer chain microspheres enriched with a large amount of capture antibodies to extend to the space under the drive of a solution, capturing and binding the colloidal gold bound with the antigen to be detected as much as possible, obviously developing the color by remaining at the T line, and capturing the residual mouse antibody at the C line by a goat anti-mouse to serve as a negative control.
Tests show that when the detection test strip provided by the invention is used for detection, the detection limit can be increased from 80.97 pg/mL to 66.99 pg/mL under the same concentration of the capture antibody, and the detection sensitivity is obviously improved.
The invention has the following advantages and effects:
(1) The surface-grafted linear polymer chain microspheres are used for the first time in the field of antigen detection of chromatographic test strips, and the Douchan effect of polyelectrolyte chain enrichment counterions is utilized, so that the surface-grafted linear polymer chain microspheres have extremely strong spatial adsorption performance, and can adsorb and concentrate a large amount of capture antibodies;
(2) The method improves the sensitivity of test strip detection by introducing the surface grafted linear polymer chain microspheres into the detection line T, does not need to change the structure of the existing chromatographic test strip, and is very convenient to apply; the test strip provided by the invention adopts the surface-grafted linear polymer chain microspheres, has low manufacturing cost, is only 0.005 to 0.002 yuan per test strip, and is convenient for social popularization.
In addition, the surface-grafted linear polymer chain microspheres provided by the invention are not limited to enriching antibodies, and can also be used for detecting other types such as viruses, biomarkers and the like. In addition, the surface grafted linear polymer chain microsphere is not limited to absorb a single antibody, and can form a compound with various antibodies to realize high-throughput detection.
Drawings
FIG. 1 is a schematic structural diagram of a surface-grafted linear polymer chain microsphere prepared in example 1 of the present invention;
FIG. 2 is a graph showing the adsorption efficiency of capture antibodies at different antibody concentrations in example 4 of the present invention;
FIG. 3 is a graph showing the adsorption amounts of the capture antibody at different antibody addition amounts in example 4 of the present invention;
FIG. 4 is a graph showing the adsorption amount of the capture antibody at different addition amounts of the surface-grafted linear polymer chain microspheres in example 4 of the present invention;
FIG. 5 is a graph showing the adsorption amounts of capture antibodies at different amounts of capture antibody added in example 5 of the present invention;
FIG. 6 is a graph comparing the results of chromatography at 0, 15 and 30 min on test strips in example 6 of the present invention;
FIG. 7 is a schematic structural diagram of a chromatographic detection test strip provided in embodiment 7 of the present invention;
FIG. 8 is a graph showing the results of detection of antigen detection solutions by the test strip with different amounts of surface-grafted linear polymer chain microspheres in example 8 of the present invention;
FIG. 9 is a comparison graph of the test strip with the surface grafted linear polymer chain microspheres and the test strip without the surface grafted linear polymer chain microspheres in example 8 of the present invention;
FIG. 10 is a graph comparing the results of the test of comparative example 1 of the present invention;
FIG. 11 is a comparison graph of the results of the test of comparative example 2 of the present invention;
FIG. 12 is a graph showing the results of detection in example 10 of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.
The technical solution of the present invention will be explained in detail below.
Example 1
This example provides a surface-grafted linear polymer chain microsphere, which is prepared by the following steps:
(1) 5.05 g initiator HMEM and 4.05 g surfactant sodium dodecyl sulfate are stirred and dissolved in 800 mL deionized water, and then the mixture is transferred into a three-neck flask; adding 40 g styrene monomer into a three-neck flask, starting mechanical stirring, pumping and filling nitrogen for 5 times, fully replacing air in the system, heating to 105 ℃ under the nitrogen atmosphere, controlling the stirring speed to be 300 revolutions per minute, carrying out polymerization reaction, and preparing polystyrene microemulsion balls;
(2) After the polymerization reaction is carried out for 8.5 hours, slowly dropwise adding 1.6 g photoinitiator HMEM into the reaction system in the nitrogen atmosphere at the final stage of emulsion polymerization reaction, and continuously reacting for 4 hours after dropwise adding is finished to obtain spherical core emulsion coated by the photoinitiator, wherein the solid content is 35%;
(3) The prepared spherical core emulsion coated by the photoinitiator and an acrylic acid monomer are mixed in a photoreactor, wherein the addition amount of the acrylic acid monomer is 80 percent of the solid content of the spherical core emulsion coated by the photoinitiator, nitrogen is pumped and filled for 5 times, vigorous stirring is carried out at normal temperature, and polymerization reaction is carried out under ultraviolet illumination, so as to prepare the surface grafted linear polymer chain microsphere.
The schematic structure of the prepared surface-grafted linear polymer chain microsphere is shown in figure 1, and the average particle size is 300-500 nm.
Example 2
This example provides a conjugate of surface-grafted linear polymer chain microspheres and capture antibodies, prepared using the surface-grafted linear polymer chain microspheres prepared in example 1, including the following steps:
40 ug the surface grafted linear polymer chain microspheres prepared in example 1 were dispersed in 1mL phosphate buffer solution (PBS buffer) with a concentration of 100 mM; and then adding a capture antibody of 400 ug, wherein the capture antibody is a monoclonal mouse-derived novel coronavirus antibody (purchased from a Fipeng organism), gently stirring for 2 hours, and adjusting the pH value to the isoelectric point of the capture antibody of 7.4 to obtain the surface-grafted linear polymer chain microsphere-capture antibody conjugate.
The obtained surface-grafted linear polymer chain microsphere-capture antibody conjugate can be used for coating a test strip detection line T.
Example 3
This example provides a conjugate of surface-grafted linear polymer chain microspheres and capture antibodies, prepared using the surface-grafted linear polymer chain microspheres prepared in example 1, including the following steps:
transferring the surface-grafted linear polymer chain microspheres prepared in example 1 of 40 ug into a low-adsorption centrifuge tube by using a liquid transfer gun, adding 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine (EDC) in an equimolar ratio to the number of carboxyl groups contained in the surface-grafted polymer chains of the surface-grafted linear polymer chain microspheres, activating at room temperature for 30 minutes, adding 40 ug capture antibody, wherein the capture antibody is a monoclonal mouse-derived novel coronavirus antibody (purchased from Fipeng biology), and linking functional groups on the polymer chains with amino acid residues of the capture antibody through chemical bonds to obtain the surface-grafted linear polymer chain microsphere-capture antibody conjugate.
The obtained surface-grafted linear polymer chain microsphere-capture antibody conjugate can be used for coating a test strip detection line T.
Example 4
In this example, the binding capacity of the surface-grafted linear polymer chain microspheres to the capture antibody by electrostatic adsorption was examined, and the capture antibody was a monoclonal mouse-derived novel coronavirus antibody (purchased from philippine).
In the first experiment, the amount of the surface grafted linear polymer chain microspheres is fixed, and the addition amount of the capture antibody is changed, wherein the experiment process comprises the following steps:
dispersing 40 ug surface grafted linear polymer chain microspheres in 50 uL PBS buffer (100 mM phosphate buffer) to prepare a surface grafted linear polymer chain microsphere solution;
the method comprises the steps of adding capture antibodies (novel monoclonal mouse-derived coronavirus antibodies produced by Fenpeng organisms) with different dosages into a surface grafted linear polymer chain microsphere solution, after the mixture is stirred for 2 hours in a mild mode, adjusting the pH value to be 7.4 of the isoelectric point of the capture antibodies, enabling the surface grafted linear polymer chain microspheres to spontaneously form soluble complexes with the capture antibodies through electrostatic interaction, and fixing a large amount of the capture antibodies in a polymer electrolyte layer.
The capture amount of the capture antibody by the surface-grafted linear polymer chain microspheres can be measured by a protein concentration test box. The concentration of the antibody is changed between 0 and 100mg/mL, and the absorption efficiency curve chart of the capture antibody is shown in figure 2; the amount of antibody added varied from 0-100ug, and the amount of capture antibody adsorbed is shown in FIG. 3. As can be seen from fig. 2, the maximum capture antibody adsorption efficiency can reach about 80%, and as can be seen from fig. 3, the absolute amount of the capture antibody adsorbed is 40 ug, that is, the surface grafted linear polymer chain microspheres can adsorb the capture antibody with the same mass as the surface grafted linear polymer chain microspheres.
Experiment two, fixing the amount of the capture antibody, changing the adding amount of the surface grafted linear polymer chain microspheres, and the experimental process is as follows:
respectively taking 10, 20, 40, 60, 80, 100, 120 and 140 ug surface grafted linear polymer chain microspheres, dispersing the surface grafted linear polymer chain microspheres in a 1.5 mL centrifugal tube containing 500 uL of PBS buffer (100 mM phosphate buffer), and preparing surface grafted linear polymer chain microsphere solutions with different concentrations;
the capture antibody 50 uL (a novel coronavirus antibody of monoclonal mouse origin produced by the Fengcong organism) with the concentration of 1.5 mg/mL is respectively added into the surface grafted linear polymer chain microsphere solutions with different concentrations, after 2 hours of mild stirring, the pH is adjusted to the isoelectric point of the capture antibody of 7.4, the surface grafted linear polymer chain microspheres can spontaneously form a soluble compound with the capture antibody through electrostatic interaction, and a large amount of the capture antibody is fixed in the polymer electrolyte layer.
The capture amount of the capture antibody by the surface-grafted linear polymer chain microspheres is measured by a protein concentration test box, and a graph of the adsorption amount of the capture antibody under different addition amounts of the surface-grafted linear polymer chain microspheres is shown in fig. 4, so that the amount of the adsorbed capture antibody is increased along with the increase of the absolute addition amount of the surface-grafted linear polymer chain microspheres.
Example 5
In this example, the binding capacity of the surface-grafted linear polymer chain microspheres to the capture antibody by chemical action was examined, and the capture antibody was a monoclonal mouse-derived novel coronavirus antibody (purchased from philippine).
The implementation process is as follows: transferring the surface grafting linear polymer chain microspheres prepared in example 1 of 40 ug into a low-adsorption centrifuge tube by using a liquid transfer gun, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) in an equimolar ratio to the number of carboxyl groups contained in the surface grafting polymer chains of the surface grafting linear polymer chain microspheres, and activating at room temperature for 30 minutes to obtain an activation liquid;
and respectively adding the capture antibodies of 20, 30, 40, 50, 60, 80 and 100ug into the activation solution, and linking the functional groups on the polymer chains and the amino acid residues of the capture antibodies through chemical bonds to obtain the surface grafted linear polymer chain microsphere-capture antibody conjugate.
The capture amount of the capture antibody by the surface-grafted linear polymer chain microspheres is measured by a protein concentration test box, and a graph of the adsorption amount of the capture antibody under different addition amounts of the capture antibody is shown in FIG. 5. As can be seen, with the increase of the addition amount of the capture antibody, the bonding of the surface-grafted linear polymer chain microspheres to the capture antibody is correspondingly increased, and the amount of the capture antibody adsorbed by the surface-grafted linear polymer chain microspheres of 40 ug is 5 ug.
Example 6
In this embodiment, the conjugate of the surface-grafted linear polymer chain microsphere and the capture antibody is used for coating the detection line T, and the immobilization effect after coating is detected.
The experimental procedure was as follows:
(1) Surface grafting linear polymer chain microsphere dyeing
Preparing a rhodamine tetrahydrofuran dye solution with the concentration of 4.9 mM, and dispersing surface grafting linear polymer chain microspheres with the concentration of 0.5 wt% in a 600 uL aqueous solution for later use; adding 100 uL dye solution into 600 uL surface grafted linear polymer chain microsphere dispersion solution, incubating and dyeing, shaking up at 1400 rpm for 30 minutes (temperature is set to 25 ℃), taking out, centrifuging and repeatedly washing for three times to complete dyeing;
(2) Preparing a dyed surface-grafted linear polymer chain microsphere-capture antibody conjugate by referring to the method in example 2, and dispersing 40 ug surface-grafted linear polymer chain microspheres in 50 uL of PBS buffer (100 mM phosphate buffer) to prepare a surface-grafted linear polymer chain microsphere solution; adding 20 ug capture antibody into the surface grafted linear polymer chain microsphere solution, wherein the capture antibody is a novel monoclonal mouse-derived coronavirus antibody (purchased from Fipeng organisms), after 2 hours of mild stirring, adjusting the pH value to the isoelectric point of the capture antibody of 7.4, forming a soluble compound with the capture antibody by spontaneous electrostatic interaction of the dyed surface grafted linear polymer chain microsphere, and fixing a large amount of capture antibody in a polymer electrolyte layer to obtain a surface grafted linear polymer chain microsphere-capture antibody conjugate;
(3) And (3) coating the test strip detection line T by using the surface grafted linear polymer chain microsphere-capture antibody combination obtained in the step (2), namely coating the obtained surface grafted linear polymer chain microsphere-capture antibody combination on the antigen chromatography test strip T line by using a scribing machine, wherein the coating width is about 0.7 cm, and drying the test strip T line.
The method for evaluating the solid-carrying effect after coating comprises the following steps:
and (3) dropwise adding a sample dilute acid solution onto the test strip prepared in the step (3), wherein the sample dilute solution is a solution of Tween-20 with the mass percentage concentration of 5%, and observing the migration degree of a dyed strip at the T line under the chromatography of the sample dilute solution, so that the loading condition of the surface grafted linear polymer chain microsphere loaded with the capture antibody at the T line is evaluated, and if the strip at the T line is not obviously migrated, the successful immobilization is proved and the immobilization effect is good.
As shown in fig. 6, which is a comparison graph of the chromatography results of the test strip prepared in this example at 0, 15, and 30 min, it can be seen from the graph that no significant deviation of the strip at the T-line is observed after 30 min of chromatography, which proves that the immobilization effect is good.
Example 7
This embodiment provides a chromatographic test strip, and the structure of the test strip is shown in fig. 7. The test strip comprises an adhesive bottom plate, a nitrocellulose membrane 3 is constructed on the adhesive bottom plate, and a detection line T (namely a T line) and a quality control line C (namely a C line) are coated on the nitrocellulose membrane 3.
The two ends of the nitrocellulose membrane 3 are respectively overlapped and provided with a gold combining pad 2 and a water absorption pad 4, and one end, far away from the nitrocellulose membrane 3, of the gold combining pad 2 is overlapped and provided with a sample pad 1. The quality control line C is arranged close to the water absorption pad 4, and the detection line T and the quality control line C are arranged at intervals and in parallel.
The length of the adhesive plastic base plate is 30 mm, and the lengths of the sample pad 1, the gold bonding pad 2, the nitrocellulose membrane 3 and the water absorption pad 4 can be adjusted and selected from 10-17 mm.
As can be seen from FIG. 7, the test strip sequentially comprises a sample pad 1, a gold seed combination pad 2, a nitrocellulose membrane 3 and a water absorption pad 4 from left to right, and the sample pad 1 and the gold seed combination pad 2, the gold seed combination pad 2 and the nitrocellulose membrane 3, and the nitrocellulose membrane 3 and the water absorption pad 4 are mutually laminated and pressed for 1-2 mm.
The gold seed combination pad 2 is sprayed and marked with antibody nano colloidal gold for specifically recognizing the antigen to be detected, and the nano colloidal gold is marked by a mouse anti-monoclonal detection antibody (a mouse anti-monoclonal capture new crown antibody produced by fenpeng biology, ltd.).
The preparation process of the mouse anti-monoclonal detection antibody labeled nano colloidal gold comprises the following steps:
(1) Preparation of colloidal gold nanoparticles AuNPs
The preparation of the colloidal gold is carried out by a conventional method, and the example provides a preparation method, which comprises the following steps:
taking a 500 mL round-bottom flask, measuring 100 g ultrapure water by using an electronic balance, adding the weighed ultrapure water into the round-bottom flask, and adjusting the temperature of an electric heating jacket to 120 ℃; after the round-bottom flask is heated for about 5 minutes, adding 1mL aqueous solution of chloroauric acid with the concentration of 1 mM; after the solution in the round-bottom flask begins to boil, the rotation speed of the rotor is increased, the sodium citrate aqueous solution with the concentration of 1mL of 1 mM is rapidly added at one time, the solution is continuously heated until the color of the solution is stable, the reaction solution is continuously heated for 5 minutes after the color of the reaction solution turns to purple red, the round-bottom flask is taken out after the heating is stopped, the round-bottom flask is placed on a vortex instrument and continuously rotated until the solution is cooled, and the colloidal gold solution containing the colloidal gold nanoparticles AuNPs is obtained.
(2) Antibody-recognizing mouse anti-monoclonal detection antibody marked on colloidal gold
And (2) taking 20mL of the colloidal gold solution obtained in the step (1), adding a recognition antibody mouse anti-monoclonal detection antibody of 50 ug, and reacting. After the reaction for 20 minutes, the gold nanoparticles with the recognition antibody marked on the surface are obtained by centrifugal separation.
Preparation of a detection line T: the T-wires on the nitrocellulose membrane 3 were coated with the surface-grafted linear polymer chain microsphere-capture antibody conjugate provided in example 2. The surface-grafted linear polymer chain microsphere-capture antibody conjugate prepared in example 2 was scribed on a nitrocellulose membrane 3 at a coating rate of 1 uL/min, with a scribe width of about 0.7 cm, to obtain a test line T.
Preparing a quality control line C: the C line on the nitrocellulose membrane 3 is coated by a goat anti-mouse multi-anti-IgG antibody, and specifically, a goat anti-mouse multi-anti-IgG antibody produced by Fengcheng biological Limited is adopted. The coating solution is scribed on the nitrocellulose membrane 3 at a coating rate of 1 uL/min, and scribed side by side with the detection line T and close to the absorbent pad 4 to obtain a detection line C.
The sample pad 1 and the absorbent pad 4 of the test strip are treated by a conventional treatment operation.
The assembly process of the chromatography detection test strip provided by the embodiment comprises the following steps: the pretreated sample pad 1, the gold combining pad 2, the nitrocellulose membrane 3 marked with the T line and the C line and the water absorption pad 4 are sequentially adhered to a plastic adhesive bottom plate from left to right, wherein the sample pad 1, the gold combining pad 2, the nitrocellulose membrane 3 and the water absorption pad 4 are sequentially laminated by 1-2 mm.
This embodiment further provides a kit, has the draw-in groove of holding the test paper strip in the kit, and the test paper strip adaptation that this embodiment made sets up in the draw-in groove, still is provided with the application of sample hole that corresponds with the sample pad on the box body and observes the hole with the testing result that nitrocellulose membrane 3 corresponds.
Example 8
In the embodiment, the influence of the contents of the grafted linear polymer chain microspheres on different surfaces on the detection sensitivity is examined when the contents of the captured antibodies in the detection line T are the same.
The detection test strip that this embodiment adopted, capture antibody concentration in the coating liquid that adopts when detection line T department coats is 1500ug/mL, and the coating volume is also the same, and detection line T department coats that the surface graft linear polymer chain microballon content is different, is 30, 60, 90ug respectively.
By adopting the test strip of the embodiment, the antigen detection solution with the antigen concentration of 125pg/mL is used as a detection object and dropped on the test strip sample pad for detection, the antigen detection solution is diluted by a Xinguan antigen produced by Fengcong biological Limited company, and the detection result is shown in fig. 8.
As can be seen from fig. 8, when the content of the captured antibody is the same and the content of the antigen in the detected antigen detection solution is the same, as the content of the surface-grafted linear polymer chain microspheres coated at the T-line is increased from 30 ug to 90ug, it can be found that the color development of the strip at the T-line is increased, which indicates that the surface-grafted linear polymer chain microspheres are added at the detection T-line, and when the detection sample liquid passes through, the polymer beads and the beads, and the chains are supported by each other and stacked upward to form a three-dimensional network structure, so that the capture efficiency of the antigen is enhanced and the detection sensitivity is improved.
The following test strips were used for the experiments: the concentration of the capture antibody in the coating solution adopted during coating at the detection line T is 1500ug/mL, the coating amount is the same, and the content of the microspheres with the linear polymer chain grafted on the coating surface at the detection line T is 0 (namely, no polymer is added in the figure) or 60ug. The antigen detection solution with the antigen concentration of 125, 250, 500 pg/mL is respectively used as a detection object and is dripped on a test strip sample pad for detection, and the antigen detection solution is diluted by a Xinguan antigen produced by Fengchen biological Limited. The detection results are shown in fig. 9.
By reading the gray scale value of the test strip shown in fig. 9, it can be known that the addition amount of the surface grafted linear polymer chain microspheres at the detection T line of the test strip is 60ug, and the antigen detection limit is significantly higher than that of the test strip without the surface grafted linear polymer chain microspheres when the concentration of the capture antibody is 1500 ug/mL.
Example 9
This example provides a method for detecting a recombinant N protein antigen stock solution using the test strip prepared in example 7. The detection process is as follows:
(1) Preparation of sample solution to be tested
Diluting a recombinant N protein antigen mother solution (a new crown antigen produced by Fenpeng biological Limited) to prepare a sample solution to be detected;
(2) Detection of sample solution to be tested
Dripping the sample solution to be detected of the 80 uL onto a sample pad 1, enabling sample liquid to flow through a gold seed combination pad 2 along with the driving of a water absorption pad 4, capturing the antigen to be detected by a recognition antibody on the surface of colloidal gold, then carrying out liquid chromatography to a detection T line, enabling surface grafting linear polymer chain microspheres enriched with a large amount of capture antibodies to extend to the space under the driving of the solution to form a three-dimensional network intercepting structure, capturing a large amount of colloidal gold bound with the antigen to be detected, remaining at the T line to realize color development enhancement, and capturing the rest of mouse anti-antibodies at a C line by goat anti-mouse antibodies to serve as negative control.
Comparative example 1
The test paper strip is characterized in that the surface grafted linear polymer chain microspheres are not added in the T-line coating solution of the ordinary test paper strip, only the captured antibody is added, and the coated captured antibody and the concentration thereof are the same as those of the test paper strip prepared in the embodiment 7.
A comparison of the results of the tests of this comparative example and example 9 is shown in FIG. 10. As can be seen from the figure, under the same concentration of the capture antibody, the detection limit of the antigen detection test strip with the T line coated with the surface grafted linear polymer chain microspheres is obviously reduced compared with the common test strip prepared by the conventional method, and is changed from 80.97 pg/mL to 66.99 pg/mL. The test paper strip can still realize accurate detection when the antigen concentration is low.
Comparative example 2
In the comparative example, three commercial neocorona antigen detection kit products are respectively adopted to carry out detection comparison with the kit prepared in the embodiment 7 of the invention.
80 uL of the dilution of the recombinant N protein antigen stock solution (New crown antigen produced by Fenpeng biological Limited) is respectively dripped onto the test strip sample pad, and after 15 minutes, the gray value of the hyperchromic strip at the T line is compared to show the quality of the detection effect. The obtained comparison of the detection results is shown in FIG. 11. The lowest detection limits of the competitive products W, A and D are 125, 250 and 100 pg/mL respectively, while the detection limit of the kit prepared in the embodiment 7 of the invention is as low as 50 pg/mL, and the detection limit is obviously reduced compared with that of the existing marketized new crown antigen detection kit product.
Example 10
In this example, the kit prepared in example 7 was used to detect inactivated new coronavirus, and the detection process was as follows:
(1) Preparation of sample solution to be tested
Taking different types of new corona inactivated strains, specifically Alpha, beta, gamma, delta, BA1, BA2 and WT (wild strains), diluting the inactivated viruses to obtain a sample solution to be tested, wherein the concentration of the diluted inactivated viruses is 6.25-0.20 TCID 50/mL;
(2) Detection of sample solution to be detected
The sample solution to be detected of the 80 uL is respectively dripped onto the sample pad 1, the sample solution flows through the gold seed combination pad 2 along with the driving of the water absorption pad 4, the virus antigen to be detected can be captured by the recognition antibody on the surface of the colloidal gold, then the sample solution flows to a detection T line along with the liquid chromatography, the surface grafting linear polymer chain microspheres enriched with a large amount of capture antibodies extend to the space under the driving of the solution to form a three-dimensional network interception structure, a large amount of colloidal gold bound with the virus antigen to be detected is captured, the color development enhancement is realized at the T line, and the rest of the mouse anti-antibody is captured by the goat anti-mouse antibody at the C line and used as a negative control.
As shown in fig. 12, it can be seen from fig. 12 that the addition of the linear polymer chain microspheres grafted on the surface of the T-line of the test strip not only improves the detection sensitivity of the test strip, but also does not affect the specific recognition of different types of viral antigens. The detection sensitivity is respectively 0.20, 0.27, 0.29, 0.84, 1.68, 1.75 and 4.62 TCID 50/mL when Wild Type (WT), BA1, gamma, BA2, delta, alpha and Beta strains are detected, compared with the detection sensitivity of the commercial neo-corona antigen detection kit product, the detection sensitivity is obviously improved, and the condition of missing detection does not occur.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Claims (15)
1. An application of a surface grafted linear polymer chain microsphere in the preparation of a test strip.
2. The use of claim 1, wherein the surface-grafted linear polymer chain microspheres are used in the preparation of a test strip detection line T.
3. Use according to claim 1 or 2, wherein the surface-grafted linear polymer chain microspheres are used as detection line T coating material.
4. The use of claim 3, wherein the surface-grafted linear polymer chain microspheres are used as a detection line T coating material after being combined with capture antibodies.
5. The use according to claim 1, wherein the surface-grafted linear polymer chain microspheres comprise polymer microspheres and linear polymer chains grafted to the surface of the polymer microspheres;
the polymer microspheres are polystyrene microspheres, micro-nano carbon spheres or nano silicon dioxide microspheres;
the linear polymer chain is polyacrylic acid, polymethacrylic acid, polyacrylamide or polystyrene sulfonate; and/or the presence of a gas in the atmosphere,
the particle size of the surface grafted linear polymer chain microsphere is 100 to 400 nm.
6. Use according to claim 5, wherein the polymeric microspheres are polystyrene microspheres; the linear polymer chain is polyacrylic acid.
7. The use of claim 4, wherein the surface-grafted linear polymer chain microspheres are bound to the capture antibodies by electrostatic or chemical interaction.
8. The detection test strip is characterized in that a detection line T is arranged on the detection test strip, the detection line T is coated with a capture antibody, and the capture antibody is bound on a surface-grafted linear polymer chain microsphere.
9. The test strip of claim 8, wherein the test line T is disposed on a nitrocellulose membrane, and a quality control line C is further disposed on the nitrocellulose membrane, and the quality control line C is coated with goat anti-mouse polyclonal IgG antibody.
10. The test strip of claim 9, wherein a gold conjugate pad and a water absorbent pad are disposed at two ends of the nitrocellulose membrane, respectively, a sample pad is disposed at one end of the gold conjugate pad away from the nitrocellulose membrane, the quality control line C is disposed near the water absorbent pad, and the test line T is spaced from and parallel to the quality control line C.
11. The method of preparing a test strip of any one of claims 8-10, comprising the steps of;
s1: preparing surface grafted linear polymer chain microspheres;
s2: preparing a conjugate of the surface grafted linear polymer chain microsphere and the capture antibody;
s3: and coating the conjugate of the surface grafted linear polymer chain microsphere and the capture antibody on a detection test strip to manufacture a detection line T.
12. The method of claim 11, wherein the step of preparing the surface-grafted microspheres with linear polymer chains comprises:
s11, polymerizing styrene serving as a monomer in an inert atmosphere to obtain polystyrene microemulsion spheres;
s12, adding a photoinitiator into a polymerization reaction system at the final stage of the polymerization reaction in the step S11 to obtain spherical core emulsion coated by the photoinitiator;
s13, polymerizing the spherical core emulsion coated by the photoinitiator and an acrylic monomer to obtain the surface grafted linear polymer chain microsphere; the using amount of the acrylic monomer is 35 to 80 percent of the solid content of the spherical core emulsion coated by the photoinitiator;
and/or the presence of a gas in the gas,
the preparation method of the conjugate of the surface grafted linear polymer chain microsphere and the capture antibody comprises the following steps:
preparation of conjugates by electrostatic interaction: mixing the surface-grafted linear polymer chain microspheres with excessive capture antibodies in a buffer solution, then adjusting the pH to the isoelectric point of the capture antibodies, and combining the surface-grafted linear polymer chain microspheres with the capture antibodies through electrostatic interaction to form a combination; alternatively, the first and second electrodes may be,
preparation of conjugates by chemical action: activating the surface grafted linear polymer chain microspheres by using a chemical cross-linking agent, and then chemically combining the activated surface grafted linear polymer chain microspheres with a capture antibody to obtain a combination; the chemical cross-linking agent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide, and the dosage of the chemical cross-linking agent is equal to the number of carboxyl groups contained in the surface grafting linear polymer chain microsphere;
and/or the presence of a gas in the atmosphere,
and coating the conjugate of the surface grafted linear polymer chain microsphere and the capture antibody on a detection test strip, drying and curing to prepare a detection line T.
13. A test kit comprising the test strip of any one of claims 8 to 10.
14. Use of the test strip of any one of claims 8 to 10 or the test kit of claim 13 for the detection of an antigen or a virus.
15. The use according to claim 14, wherein the antigen is a recombinant N protein antigen and/or the virus is Alpha, beta, gamma, delta, BA1, BA2, WT wild strain.
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