CN115248308B

CN115248308B - Application of poly-L-dopa nano-particles in preparation of immunochromatography detection test strip

Info

Publication number: CN115248308B
Application number: CN202210775176.9A
Authority: CN
Inventors: 唐建斌; 何康松; 叶雅冰; 王信
Original assignee: Hangzhou Moju Biotechnology Co ltd; Zhejiang University ZJU
Current assignee: Hangzhou Moju Biotechnology Co ltd; Zhejiang University ZJU
Priority date: 2022-07-01
Filing date: 2022-07-01
Publication date: 2024-05-31
Anticipated expiration: 2042-07-01
Also published as: CN115248308A

Abstract

The invention discloses an application of poly-L-dopa nano-particles in preparing an immunochromatography detection test strip, and belongs to the technical field of in-vitro diagnostic reagents. The immunochromatography detection test strip comprises a binding pad, wherein the binding pad is coated with a poly-L-dopa nanoparticle-antibody/antigen complex, and the complex is prepared by coupling carboxyl groups on the surface of the poly-L-dopa nanoparticle with an antibody or an antigen. The carboxyl group on the surface of the poly-L-dopa nanoparticle is covalently combined with protein to form a stable compound, the biological activity of the protein is not affected, and the poly-L-dopa nanoparticle is aggregated to generate macroscopic black, and compared with the red of colloidal gold, the poly-L-dopa nanoparticle has higher color rendering index, so that the poly-L-dopa nanoparticle can be used as a tracer marker for antigen-antibody reaction.

Description

Application of poly-L-dopa nano-particles in preparation of immunochromatography detection test strip

Technical Field

The invention relates to the technical field of in-vitro diagnostic reagents, in particular to application of poly-L-dopa nano-particles as an immune marker in preparation of an immune chromatography detection test strip.

Background

The principle of the labeling immune technology is to use fluorescein, radioisotope, enzyme and the like to label an antibody or an antigen, and detect the corresponding antigen or antibody through antigen-antibody reaction. The specificity of the antigen-antibody reaction and the amplification effect of the marker ensure the specificity and sensitivity of the marker immune technique.

The immunochromatography detection technology (Lateral Flow Immunoassay, LFIA) is an immunological detection technology developed at the beginning of the 80 th century, and the working principle is as follows: one specific antibody against the antigen to be tested is immobilized in a strip on a nitrocellulose membrane (NC) as a detection zone (T zone), and the other specific antibody is conjugated with a label and immobilized on a binding pad. When the sample of the object to be detected is added to the sample pad at one end of the test strip, the sample moves forward through capillary action, the marker reagent on the binding pad is dissolved and then reacts with each other, and then the sample moves to the T area, the conjugate of the object to be detected and the marker reagent is combined with the antibody corresponding to the T area to be trapped, and the conjugate is gathered in the T area to form a colored strip.

The performance of the label in the immunolabeling technique is a key factor affecting the accuracy and sensitivity of the detection result. Colloidal gold is the most commonly used marker in immunochromatography, colloidal gold is hydrosol of Jin Shanzhi (Au), and colloidal gold particles have a strong adsorption function on proteins, do not destroy the biological activity of the proteins, and can be non-covalently combined with proteins and the like to form the colloidal gold marker. The colloidal gold particles can be colored, and thus are used as tracer markers for antigen-antibody reactions. The colloidal gold immunochromatography is generally qualitative or semi-quantitative detection, is rapid and convenient, and has rapid feedback, but insufficient stability and poor sensitivity of detection results. In view of this, some luminescent materials including fluorescence, up-conversion phosphorescence, chemiluminescent materials, etc. and magnetic materials have been developed successively, so that the detection sensitivity is greatly improved, but the luminescent material markers often need precise instruments during detection, and the operation steps are complex, consume long time, and are not suitable for instant detection and field application.

In view of the characteristics of convenient and visual operation and rapid feedback of the colloidal gold immunochromatographic test strip, the detection test strip marked by using the colloidal gold is still a mainstream commodity currently applied to on-site instant detection, however, the chloroauric acid for preparing the colloidal gold is expensive, and the detection test strip circulates in the market and has the problem of heavy metal pollution.

Therefore, developing a label that can replace colloidal gold, is lower in manufacturing cost, and has excellent color rendering properties for use in immunochromatography test strips is a problem that needs to be solved by those skilled in the art.

Disclosure of Invention

The invention aims to provide a novel nanoparticle for labeling an antibody or an antigen to be applied to immunochromatography detection, so that the manufacturing cost of the test paper is reduced, the detection sensitivity of the test paper is improved, and the possibility is further provided for semi-quantitative or quantitative detection of the test paper.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention provides application of poly-L-dopa nano-particles as markers in preparation of immunochromatography detection test strips.

Furthermore, the immunochromatography detection test strip comprises a binding pad, wherein the binding pad is coated with a poly-L-dopa nanoparticle-antibody/antigen complex, and the complex is prepared by coupling carboxyl groups on the surface of the poly-L-dopa nanoparticle with an antibody or an antigen.

The immunochromatography detection test strip consists of a PVC bottom plate, and a sample pad, a bonding pad, a chromatographic membrane and a water absorption pad which are adhered on the PVC bottom plate and sequentially paved along the chromatography direction, wherein the sample pad is partially overlapped on the bonding pad, the bonding pad is partially overlapped on one side of the chromatographic membrane, and the water absorption pad is partially overlapped on the other side of the chromatographic membrane. The invention couples the poly-L-dopa nano-particles with the labeled antibody or antigen, and coats the poly-L-dopa nano-particles on a binding pad, thereby providing a novel immunochromatography detection test strip.

The poly-L-dopa nano-particles are nano-particles formed by self-polymerization of the L-dopa monomers, a large number of carboxyl groups exist on the surfaces of the poly-L-dopa nano-particles, and can be covalently combined with protein to form a stable complex, and meanwhile, the covalent combination does not damage the biological activity of the protein. In addition, the poly-L-dopa nanoparticle aggregation produces a macroscopic black color, and therefore, the poly-L-dopa nanoparticle can be used as a tracer marker for antigen-antibody reaction. The research of the invention shows that aggregation of the poly-L-nanometer particles with large particle size can generate high-strength black so as to remarkably improve the detection sensitivity.

Further, the preparation method of the poly-L-dopa nanoparticle-antibody/antigen complex comprises the following steps: firstly, activating carboxyl groups on the surfaces of poly-L-dopa nano particles by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), then adding an antibody or antigen for coupling, and then adding a blocking solution for blocking to obtain the compound.

Further, the preparation method of the poly-L-dopa nanoparticle comprises the following steps: the levodopa monomer is mixed with a metal ion complexing agent and then reacts for 12 to 24 hours at the temperature of between 70 and 80 ℃ to prepare the coating.

In the preparation process, the levodopa monomer and the metal ion complexing agent are mixed in water, and under the complexing action of metal ions, the self-polymerization process of the levodopa monomer is accelerated. The metal ion complexing agent includes, but is not limited to, nickel ions, ferric ions, magnesium ions, calcium ions, or cobalt ions.

Preferably, the concentration of the levodopa monomer in the reaction system is 0.5-1 mg/mL.

Preferably, the metal ion complexing agent is nickel acetate.

Research shows that the particle size distribution (PDI) and the particle size of the poly-L-dopa nano particles influence the subsequent detection performance, and the larger the particle size, the better the color development effect of the nano particles under the same target substance concentration. Preferably, the particle size of the poly-L-dopa nano particle is 150-350 nm.

In the preparation process of the poly-L-dopa nano-particles, the higher the metal ion content in the reaction system is, the larger the particle size of the prepared nano-particles is relatively. Preferably, the molar ratio of the levodopa monomer to the metal ion complexing agent is 20-112: 1.

Preferably, the molar ratio of the levodopa monomer to the nickel acetate is 25-60: 1, a step of; the reaction temperature was 75℃and the reaction time was 15 hours.

In the invention, the antibody/antigen coupled with the poly-L-dopa nano-particles is the antibody/antigen specifically combined with the target substance to be detected, and the corresponding antibody or antigen can be selected according to the target substance to be detected, so that the immunochromatography detection test strip for detecting the target substance is prepared.

When the bonding pad is prepared, the poly-L-dopa nano-particles coupled with specific antibodies/antigens are sprayed on the pretreated bonding pad, and the bonding pad is prepared after drying. Preferably, the bonding pad is made of an ostone 8964 glass fiber. The bonding pad treatment solution is a mixed solution of phosphate buffer solution, a stabilizing agent and a re-dissolving agent, and the pH value is 7-8.

When the chromatographic membrane is prepared, the corresponding antibody and the double antibody are selected to be diluted in the coating diluent, a detection line and a quality control line are sequentially arranged at intervals in parallel along the chromatographic direction, and the chromatographic membrane is prepared after drying. Preferably, the chromatographic membrane is a nitrocellulose membrane. The coating diluent is a mixed solution of phosphate buffer and sucrose, and the pH value is 7.4.

Preferably, the sample pad adopts an osetron 8964 glass fiber, and the sample pad treatment solution is a mixed solution of Tris buffer solution, a detergent, a sealing agent and a preservative, and the pH value is 7-8.

Specifically, the invention provides a novel inflammation marker procalcitonin (procalcitonin, PCT) detection test strip, which has good sensitivity in distinguishing bacterial infection and virus infection and has great guiding value for antibiotic treatment monitoring. PCT is the best indicator in the assisted diagnosis of severe infections and sepsis, and can also be an early marker of sepsis.

The PCT detection test strip comprises a sample pad, a binding pad, a chromatographic membrane and a water absorption pad which are sequentially arranged along the chromatographic direction, wherein detection microspheres are coated on the binding pad, and the detection microspheres are poly-L-dopa nanoparticles with PCT labeled antibodies coated on the surfaces.

Wherein, the preparation of the bonding pad comprises: firstly, preparing poly-L-dopa nano-particles, coupling carboxyl groups existing on the poly-L-dopa nano-particles with PCT labeled antibodies to obtain poly-L-dopa nano-particles-PCT labeled antibody complexes, and coating the poly-L-dopa nano-particles-PCT labeled antibody complexes on a binding pad.

The preparation of the chromatographic membrane comprises the following steps: respectively diluting PCT coated antibody and goat anti-mouse IgG double antibody into 2mg/mL and 1mg/mL coated solution by using coated diluent; and scribing lines at intervals of 5mm on the nitrocellulose membrane stuck on the PVC bottom plate respectively to obtain a detection line and a quality control line, wherein the scribing parameters are 1 mu L/cm, and the nitrocellulose membrane is dried for 16-48 hours in an environment with the relative humidity of 30-50% and the temperature of 37 ℃ after the scribing is finished.

Further, the preparation method of the novel PCT detection test strip comprises the following steps:

(1) Preparing a bonding pad: firstly preparing poly-L-dopa nano particles by using a levodopa monomer and nickel acetate, then washing the poly-L-dopa nano particles by using an MES buffer solution, adjusting the pH of a system to 5.5-6.7, coupling with PCT labeled antibodies by using EDC and NHS activated microsphere surface carboxyl groups, and then performing sealing treatment by using casein solution to prepare the poly-L-dopa nano particle-PCT labeled antibody compound; finally, spraying the poly-levodopa nanoparticle-PCT labeled antibody compound on the pretreated bonding pad;

(2) Preparing a chromatographic membrane: PCT coated antibody is sprayed on a nitrocellulose membrane by adopting a spraying point quantity of 1 mu L/cm to prepare a detection line, and sheep anti-mouse lgG double antibody is sprayed on the nitrocellulose membrane by adopting a spraying point quantity of 1 mu L/cm to prepare a quality control line; the detection line is parallel to the quality control line and is 5mm away;

(3) Assembling a test strip: and sequentially paving a sample pad, a bonding pad, a chromatographic membrane and a water absorption pad on the PVC bottom plate along a chromatography method, wherein the sample pad is partially overlapped on the bonding pad, the bonding pad is partially overlapped on one side of the chromatographic membrane, and the water absorption pad is partially overlapped on the other side of the chromatographic membrane to obtain the reagent strip.

Preferably, the concentration of PCT labeled antibody used is 0.13mg/mL, the concentration of PCT coated antibody used is 2mg/mL, and the concentration of goat anti-mouse IgG diabody used is 1mg/mL.

Furthermore, the invention also provides a method for quantitatively detecting PCT by using the novel PCT detection test strip, specifically, a sample to be detected is taken and added into PBS buffer solution, after 10s of mixing, sample adding chromatography is carried out, the gray ratio of a detection line to a quality control line is determined, and the concentration value of PCT in the sample is calculated according to a standard curve.

The invention also provides a novel Human Chorionic Gonadotrophin (HCG) detection test strip, HCG is glycoprotein secreted by trophoblast cells of a pregnant woman placenta, and whether pregnancy occurs can be judged by detecting the HCG level. The HCG detection test strip comprises a sample pad, a binding pad, a chromatographic membrane and a water absorption pad which are sequentially arranged along the chromatographic direction, wherein the binding pad is coated with a beta-HCG monoclonal antibody marked by poly-levodopa nano-particles.

The preparation method of the novel HCG detection test strip comprises the following steps:

(1) Preparing a bonding pad: firstly preparing poly-L-dopa nano-particles by using a levodopa monomer and nickel acetate, then washing the poly-L-dopa nano-particles by using a PBS buffer solution, regulating the pH value of a system to 7-8, coupling with a beta-HCG monoclonal antibody by using EDC and NHS activated microsphere surface carboxyl, and then performing sealing treatment by using a casein solution to prepare the poly-L-dopa nano-particle-beta-HCG monoclonal antibody compound; finally, spraying the poly-L-dopa nanoparticle-beta-HCG monoclonal antibody compound on the pretreated bonding pad;

(2) Preparing a chromatographic membrane: spraying an alpha-polyclonal antibody on a nitrocellulose membrane by adopting a spraying point quantity of 1 mu L/cm to prepare a detection line, and spraying a goat anti-mouse IgG double antibody on the nitrocellulose membrane by adopting a spraying point quantity of 1 mu L/cm to prepare a quality control line; the detection line is parallel to the quality control line and is 5 mm away;

Preferably, the concentration of the adopted beta-HCG monoclonal antibody is 0.2mg/mL, the concentration of the adopted alpha-polyclonal antibody is 1mg/mL, and the concentration of the adopted goat anti-mouse IgG double antibody is 0.5mg/mL.

The width of the novel HCG detection test strip prepared in the invention is 4mm, the amount of the coated alpha-polyclonal antibody at the detection line is jointly determined by the concentration of the alpha-polyclonal antibody and the spraying point amount of the alpha-polyclonal antibody, and the amount of the coated goat anti-mouse IgG double antibody at the quality control line is jointly determined by the concentration of the goat anti-mouse IgG double antibody and the spraying point amount of the goat anti-mouse IgG double antibody.

The invention has the beneficial effects that:

(1) The poly-L-dopa nano particle surface applied by the invention has a large number of carboxyl groups, and can be used for coupling with an antibody or an antigen to obtain a more stable immune complex.

(2) Compared with the existing colloidal gold technology, the preparation method has the advantages that the price of the levodopa monomer for preparing the poly-levodopa nano-particles is about one percent of that of chloroauric acid, and the cost advantage is obvious; the aggregation of the poly-L-dopa nano-particles produces macroscopic black, has higher color rendering compared with the red of colloidal gold, and is suitable for macroscopic observation or semi-quantitative and quantitative analysis.

(3) The preparation process of the poly-L-dopa nano-particles is simple and convenient.

Drawings

FIG. 1 is a schematic diagram of a test strip according to the present invention.

Fig. 2 is a schematic structural diagram of a test card according to the present invention.

FIG. 3 is a schematic representation of the reaction scheme and entity for the preparation of poly-L-dopa nanoparticles in example 1.

Fig. 4 is a graph of DLS particle size analysis of the poly-l-dopa nanoparticle of example 1.

FIG. 5 is a graph of DLS particle size analysis of poly L-dopa nanoparticle- β -HCG monoclonal antibody immunocomplexes of example 1.

FIG. 6 is a verification result illustration of the novel HCG test strip in example 1.

FIG. 7 is a graph showing the results of detecting beta-HCG antigen solutions of different concentrations using the novel HCG test strip prepared in example 1.

FIG. 8 shows the detection results of novel HCG detection test strips prepared from poly (L-dopa) nanoparticles with different particle sizes in example 1.

Fig. 9 is a graph of DLS particle size analysis of the poly-l-dopa nanoparticles of example 2.

FIG. 10 is a graph of DLS particle size analysis of the poly L-dopa nanoparticle-PCT labeled antibody complex of example 2.

FIG. 11 is a standard curve of PCT quantitative test strip in example 2 for detecting PCT standard solution.

FIG. 12 is a comparative chart of PCT quantitative detection test strips and colloidal gold detection test strips for detecting PCT standard solution in example 2, wherein the first behavior of the PCT quantitative detection test strip prepared in example 2 and the second behavior of the colloidal gold detection test strip are PCT antigen solutions with detection concentrations of 0.1 mug/L, 5 mug/L, 10 mug/L and 31 mug/L in sequence from left to right.

Detailed Description

The invention will be further illustrated with reference to specific examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

In the following examples, the β -HCG monoclonal antibody, the α -polyclonal antibody and the goat anti-mouse IgG diabodies were purchased from Hua Yangzheng Dragon Biochemical research laboratory as colorless liquids.

PCT-labeled antibodies, PCT-coated antibodies and goat anti-mouse IgG diabodies were all purchased from blue torch biotechnology (hangzhou) as colorless liquids.

Nitrocellulose membranes were purchased from Sidoris and were white in color, uniform in texture, flat in surface, free of imperfections, and neat in membrane edges, free of distortion.

The auslong 8964 glass fiber is purchased from Shanghai gold mark biotechnology limited company, and has uniform and compact fiber structure distribution, flatness and no dirty mark, dirt and dust on the peripheral edge.

Levodopa was purchased from mikrin.

Nickel acetate tetrahydrate was purchased from Guangdong Guanghua technology Co.

The english abbreviations for the compounds referred to in the examples are as follows:

EDC:1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; NHS: n-hydroxysuccinimide; MES:2- (N-morpholino) ethanesulfonic acid.

Example 1HCG test strip

1. Composition of novel HCG detection test strip

As shown in FIG. 1, the immunochromatographic test strip comprises a sample pad 1, a polymer nanoparticle-antibody coated binding pad 2, a chromatographic membrane 3, and a detection line 4 and a quality control line 5 sequentially arranged on the membrane along the chromatographic direction, and finally a water absorption pad 6, which are sequentially arranged on a PVC plate 7 along the chromatographic direction. The sample pad 2 and the water absorption pad 6 are respectively arranged at two ends of the PVC plate 7, the chromatographic membrane 3 is arranged in the middle of the PVC plate 7, and a binding pad 2 coated by polymer nano particles-antibodies is arranged between the chromatographic membrane 3 and the sample pad 1; one end of the polymer nanoparticle-antibody coated binding pad 2 is overlapped with the sample pad 1, and the other end is overlapped with the chromatographic carrier 3; and a detection line 4 and a quality control line 5 are sequentially arranged on the chromatographic membrane 3 at intervals in parallel along the chromatographic direction.

The binding pad 2 of the HCG detection test strip of this embodiment is coated with a β -HCG monoclonal antibody labeled with poly-levodopa nanoparticles, and the binding pad is oslong 8964 glass fiber. The detection line 4 is coated with an alpha-polyclonal antibody, and the quality control line 5 is coated with a goat anti-mouse IgG diabody. The detection line 4 and the quality control line 5 on the chromatographic membrane 3 are kept in parallel and are 5mm apart, and the chromatographic membrane is a nitrocellulose membrane.

The test paper strip is arranged in the detection card, the test paper strip is arranged in a plastic shell formed by inserting a plastic upper shell and a plastic lower shell, and the detection card is obtained, and the plastic upper shell is provided with two open holes, a sample adding hole 8 and an observation window 9, as shown in fig. 2.

2. Preparation method of novel HCG detection test strip

Step 1, preparing poly-L-dopa nano particles

120Mg of levodopa and 2.7mg of nickel acetate are mixed in 120mL of ultrapure water, stirred at 75 ℃ for reaction for 15 hours, centrifuged to obtain poly-levodopa nano-particles, and the poly-levodopa nano-particles are resuspended in water to have a solid content of about 3%, wherein the reaction formula and polymer are as shown in figure 3.

FIG. 4 is a graph of particle size analysis of the obtained poly (L-dopa) nanoparticles, which have an average particle size of 203.7nm and a PDI of 0.002.

Step 2, conjugation of PolyLevodopa nanoparticles with beta-HCG monoclonal antibody

And (3) regulating the pH value of the system to 7-8 by using a PBS buffer solution, enabling the surface carboxyl groups of the microspheres to be coupled with the monoclonal antibodies by using EDC and NHS, then performing blocking treatment by using a 10% casein solution, and finally storing the immune complex in a specially prepared preservation solution.

FIG. 5 is a graph of DLS particle diameter analysis of immune complex, which has an average particle diameter of 256.6nm and a PDI of 0.047.

Step 3, pretreatment of sample pad and bond pad

The sample pad treatment solution was prepared according to the following formulation: 0.04mol/L Tris, 4g/L Pvp, 3g/L T-Casein, 4g/L S, 1% Tween20, 0.8g/L PC-300 and ultrapure water are taken as solvents, the pH is regulated to 7-8, then the sample pad is placed in a treatment liquid for 3 seconds, and then the sample pad is placed in a baking oven at 37 ℃ for drying 24 hours for standby.

The bonding pad treatment liquid is prepared according to the following proportion: the pH is adjusted to 7-8 by 15% sucrose, 0.5% sodium caseinate, 0.5% Pvp30, 0.02% PC-300 and 84% PBS (solvent), then the bonding pad is immersed in the treatment liquid for 3 seconds, and then dried in a baking oven at 37 ℃ for 24 hours for later use.

The labeled poly-L-dopa nanoparticle-beta-HCG monoclonal antibody immune complex is coated on the pretreated binding pad.

Step 4, pretreatment of chromatographic membranes

Preparing a scribing diluent: 4g/L sodium chloride, 0.2g/L potassium chloride, 1.44g/L disodium hydrogen phosphate, 0.28g/L potassium dihydrogen phosphate, and 0.2g/L preservative PC-300% sucrose pH is adjusted to 7.4, and ultrapure water is used as a solvent.

And diluting the alpha-polyclonal antibody and the goat anti-mouse IgG double antibody to 1mg/mL and 0.5mg/mL respectively by using a membrane drawing diluent, respectively spraying the alpha-polyclonal antibody and the goat anti-mouse IgG double antibody on a detection line and a quality control line according to the spraying amount of 1uL/cm, and then drying for 24 hours at 37 ℃ for later use.

Step 5, assembling the test strip

The sample pad 1, the bonding pad 2, the chromatographic membrane 3 and the water absorbing paper 6 are sequentially paved on the PVC bottom plate 7 along a chromatography method, the sample pad 1 is partially overlapped on the bonding pad 2, the bonding pad 2 is partially overlapped on one side of the analysis membrane 3, and the water absorbing paper 6 is partially overlapped on the other side of the chromatographic membrane 3, so that the reagent strip is obtained.

The width of the novel HCG detection test strip prepared in the embodiment is 4mm, and the liquid moving speed is not lower than 1cm/min.

3. Application method of novel HCG detection test strip

And a step a, recovering the novel HCG detection test strip, the antigen diluent and the sample to room temperature, wherein the antigen diluent can be commercial antigen diluent or PBS buffer solution, and the sample is standard beta-HCG antigen solution or medical urine sample.

Step b, taking 50uL diluted samples and directly adding the samples into sample adding holes on a sample pad;

C, grinding and judging the result within 10-20 min;

The detection results are explained as follows:

positive: the detection line and the quality control line are both developed, which indicates that the sample contains the beta-HCG antigen;

negative: the detection line does not develop color, and the quality control line develops color, which indicates that the sample does not contain beta-HCG antigen;

Invalidation: the quality control line does not develop color, which indicates that the novel HCG detection test strip is invalid and the sample detection result is also invalid. See fig. 6.

4. Verification of novel HCG detection test strip

And (3) preparing HCG standard solution. 10. Mu.l of 2500IU per ml of standard beta-HCG antigen solution was taken, 490. Mu.l of ultrapure water was added, and diluted to 50IU per ml. The beta-HCG antigen solution of 50IU per milliliter is diluted to 0.001IU per milliliter, 0.01IU per milliliter, 0.1IU per milliliter, 1IU per milliliter and 10IU per milliliter respectively for later use.

The results obtained after 50. Mu.L of prepared beta-HCG antigen solutions with different concentrations are respectively dripped into sample holes and waiting for 20min are shown in FIG. 7, and the detection limit of the novel HCG detection test strip can reach 1IU/L, and the novel HCG detection test strip shows better sensitivity than that of colloidal gold. The specific results are shown in Table 1.

TABLE 1

Meanwhile, the color development degree of the nano particles (170 nm, 240nm and 320 nm) with different particle sizes under the same concentration is also verified, and as shown in fig. 8, the result shows that the color development effect of the nano particles with larger particle sizes is better under the same antigen concentration.

Example 2PCT quantitative detection test paper strip

1. Composition of novel quantitative detection test strip for inflammatory marker PCT and composition of detection card

The composition structure of the PCT quantitative detection test strip for the inflammatory marker in the embodiment is the same as that shown in fig. 1, specifically, detection microspheres are coated on the test strip binding pad 2, and the detection microspheres are polymer nanoparticles coated with PCT labeled antibodies on the surfaces; the chromatographic membrane 3 is sequentially provided with a detection line 4 and a quality control line 5 at intervals in parallel along the chromatographic direction, the detection line 4 is coated with PCT specific antibodies, and the quality control line 5 is coated with goat anti-mouse IgG double antibodies.

2. Preparation method of novel quantitative detection test strip for inflammatory marker PCT

The method specifically comprises the following steps:

1. Preparation of Polylevodopa nanoparticles

200Mg of levodopa and 10mg of nickel acetate were mixed in 200mL of ultrapure water, and reacted at 75℃with stirring for 15 hours. After the reaction is finished, the concentrated poly-L-dopa nano suspension is obtained by centrifugal cleaning for three times, and the solid content is 3%.

FIG. 9 is a graph of particle size analysis of the obtained poly (L-dopa) nanoparticles, which have a particle size of 251.3nm and a PDI of 0.149.

2. Chromatographic membrane treatment

Preparing a coating liquid, wherein a PCT coating antibody and a goat anti-mouse IgG double antibody are respectively diluted to 2mg/mL and 1mg/mL by a membrane-drawing diluent (pH 7.0, the formula is shown in Table 2); and (3) drawing a film by using a film drawing instrument, wherein the film drawing parameter is 1 mu L/cm, drawing 1 quality control line (C line), drawing 1 detection line (T line), and drying for 16-48h under the environment of the relative humidity of 30-50% and the temperature of 37 ℃ after finishing drawing the film.

Table 2 film-drawing diluent formulation

3. Marking

Cleaning: taking 1mL of poly-L-dopa nanoparticle suspension (solid content is 3%), centrifuging 17000g to remove supernatant, adding 1mL of MES buffer (100 mM, pH 6.0), ultrasonically cleaning, centrifuging to remove supernatant, and cleaning three times;

Activating: adding 3. Mu.L NHS (20 mg/mL), reacting at room temperature for 3min, adding 5. Mu.L EDC (20 mg/mL), reacting at room temperature for 20min, centrifuging 17000g to remove supernatant, adding 900. Mu.L MES (100 mM, pH 6.0) and 100. Mu.L ultra-pure water, and performing ultrasonic dispersion;

coupling: adding 58 mu L of PCT labeled antibody, reacting for 2h at room temperature, centrifuging 17000g to remove supernatant;

Closing: adding 1mL of sealing solution (pH 8.0, formula shown in Table 3) into the centrifugal precipitate, reacting for 2h at room temperature, centrifuging to remove supernatant;

Table 3 sealing liquid formulation

Name of the name	Concentration of
		Ethanolamine	25mM
Casein protein	10％

And (3) preserving: 1mL of a preservation buffer (pH 7.2, formula shown in Table 4) was added, and the mixture was subjected to ultrasonic dispersion to obtain a suspension of poly-L-dopa nanoparticle-PCT labeled antibody complex.

FIG. 10 is a graph of a DLS particle size analysis of a poly-L-dopa nanoparticle-PCT labeled antibody complex having an average particle size of 343.7nm and a PDI of 0.257.

Table 4 preservation solution formulation

Name of the name	Concentration of
		HEPES	25mM
Casein protein	10g/L
		PVP	2g/L
NaN₃	0.2g/L

Pretreatment of the bonding pad: the bonding pad was immersed in the bonding pad treatment solution (pH 7-8, formulation shown in Table 5) for 3s, and then placed in a vacuum oven for vacuum drying for 1h for use.

Table 5 formulation of pad treatment fluid

Marking: spraying the pretreated bonding pad of the suspension by a metal spraying instrument, wherein the bonding pad is an ostone 8964 glass fiber material, and placing the bonding pad in a vacuum drying oven for vacuum drying for 1h for later use.

4. Sample pad pretreatment

The sample pad was immersed in the sample pad treatment solution (pH 7-8, formulation shown in Table 6) for 3s, and then placed in a vacuum oven for vacuum drying for 1h for use.

Table 6 sample pad treatment fluid formulation

Name of the name	Concentration of
		Tris	0.04mol/L
PVP10	4g/L
		T-Casein	3g/L
S9	4g/L
		PC-300	0.8g/L
Tween20	1％

5. Assembled detection card

Assembling a test strip: and (3) attaching a nitrocellulose membrane to the middle part of a PVC (polyvinyl chloride) bottom plate, attaching a dried bonding pad to the PVC bottom plate, overlapping the bonding pad and the nitrocellulose membrane by 1-2mm, attaching a dried sample pad to the PVC bottom plate, overlapping the sample pad and the bonding pad by 1-2mm, attaching absorbent paper to the other end of the PVC bottom plate, overlapping the absorbent paper and the nitrocellulose membrane by 1-2mm, cutting the attached large plate into small strips of 4mm by a strip cutting machine, and assembling the test strip in a plastic shell formed by inserting a plastic upper shell and a plastic lower shell.

The test strip prepared in this example has a width of 4mm and a liquid movement speed of not less than 1cm/min.

3. Application method of novel quantitative detection test strip for inflammatory marker PCT

Step a, the novel PCT detection test strip, PBS buffer and serum sample are restored to room temperature.

Step b, taking 80 mu L of a mixed liquid sample of PBS buffer solution and serum, and directly adding the mixed liquid sample into a sample adding hole on a detection card;

c, inserting a detection test strip into a detection groove of the analyzer, and placing for 15-20min;

The detection results are explained as follows:

positive: the detection line and the quality control line are both developed, which indicates that the sample contains PCT antigen;

Negative: the detection line does not develop color, and the quality control line develops color, which indicates that the sample does not contain PCT antigen;

Invalidation: the quality control line does not develop color, which indicates that the novel PCT detection test strip is invalid and the sample detection result is also invalid.

4. PCT is detected by the test strip prepared in the embodiment

Preparing: PCT standard solutions of different concentrations were prepared, including 0.1. Mu.g/L, 0.6. Mu.g/L, 1. Mu.g/L, 10. Mu.g/L, 40. Mu.g/L, 60. Mu.g/L, 80. Mu.g/L, 100. Mu.g/L, in three replicates for each concentration. After chromatographic reaction for 15-20min, detecting gray signals at the detection line and the control line by adopting an instrument analysis device to obtain a relative gray value T/C, taking the concentration value of PCT standard liquid as an abscissa and taking T/C as an ordinate, establishing an equation and fitting into a standard curve.

As can be seen from the standard curve of fig. 11, the T/C relative gray value shows a good linear correlation with the concentration value of PCT standard solution, and the correlation coefficient R ² = 0.9919, and thus PCT concentration in a sample can be quantified by the standard curve.

PCT antigen solutions of 0.1 mug/L, 5 mug/L, 10 mug/L and 31 mug/L are respectively dripped into sample holes, and the results obtained after waiting for 15-20min are shown in FIG. 12, so that the PCT quantitative detection test strip for the novel inflammation marker can be seen to show better sensitivity than that of colloidal gold (purchased from Shandong wheat Tian Shengwu).

The above embodiments are merely preferred embodiments of the present invention, and not all. Based on the examples in the embodiments, those skilled in the art can obtain other examples without making any inventive effort, which fall within the scope of the invention.

Claims

1. The application of the poly-L-dopa nano-particles as a marker in the preparation of immunochromatography detection test strips is characterized in that the preparation method of the poly-L-dopa nano-particles comprises the following steps: mixing levodopa monomer and metal ion complexing agent, and reacting for 12-24 hours at 70-80 ℃ to obtain the compound; the particle size of the poly-L-dopa nano particle is 150-350 nm.

2. The use according to claim 1, wherein the immunochromatographic test strip comprises a binding pad coated with a poly-levodopa nanoparticle-antibody/antigen complex prepared by coupling carboxyl groups on the surface of poly-levodopa nanoparticles with an antibody or antigen.

3. The use according to claim 2, wherein the preparation method of the poly-levodopa nanoparticle-antibody/antigen complex comprises: the EDC and NHS are used for activating carboxyl on the surface of the poly-L-dopa nano particle, then an antibody or antigen is added for coupling, and then a blocking solution is added for blocking, so that the compound is prepared.

4. The use according to claim 1, wherein the molar ratio of levodopa monomer to metal ion complexing agent is 20 to 112:1, a step of; the metal ion complexing agent is nickel acetate.

5. The use according to claim 4, wherein the molar ratio of levodopa monomer to nickel acetate is 25 to 60:1, a step of; the reaction temperature was 75℃and the reaction time was 15 hours.

6. The inflammation marker procalcitonin detection test strip comprises a sample pad, a combination pad, a chromatographic membrane and a water absorption pad which are sequentially arranged along the chromatographic direction, and is characterized in that detection microspheres are coated on the combination pad, and the detection microspheres are poly-levodopa nanoparticles of which the surfaces are coated with procalcitonin labeled antibodies; the preparation method of the poly-L-dopa nano-particle comprises the following steps: mixing levodopa monomer and metal ion complexing agent, and reacting for 12-24 hours at 70-80 ℃ to obtain the compound; the particle size of the poly-L-dopa nano particle is 150-350 nm.

7. The method for preparing the test strip for detecting procalcitonin, an inflammation marker, according to claim 6, comprising the steps of:

(2) Preparing a chromatographic membrane: PCT coated antibody is sprayed on a nitrocellulose membrane by adopting a spraying point amount of 1 mu L/cm to prepare a detection line, and sheep anti-mouse IgG double antibody is sprayed on the nitrocellulose membrane by adopting a spraying point amount of 1 mu L/cm to prepare a quality control line; the detection line is parallel to the quality control line and is 5 mm away;

8. The human chorionic gonadotrophin detection test strip comprises a sample pad, a binding pad, a chromatographic membrane and a water absorption pad which are sequentially arranged along the chromatographic direction, and is characterized in that the binding pad is coated with a beta-HCG monoclonal antibody marked by poly-levodopa nano-particles; the preparation method of the poly-L-dopa nano-particle comprises the following steps: mixing levodopa monomer and metal ion complexing agent, and reacting for 12-24 hours at 70-80 ℃ to obtain the compound; the particle size of the poly-L-dopa nano particle is 150-350 nm.

9. The method of preparing a test strip for detecting human chorionic gonadotrophin according to claim 8, comprising the steps of: