Chromatographic test paper for detecting VEGF in fingertip blood, preparation method and application thereof

Abstract

This application relates to the field of biological detection and specifically discloses a chromatographic test strip for detecting VEGF in fingertip blood, its preparation method, and its application. The chromatographic test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorption pad, which are sequentially overlapped. The conjugate pad is coated with modified sporopollenin microspheres, and the surface of the modified sporopollenin microspheres is labeled with an anti-VEGF monoclonal antibody and a rabbit anti-mouse antibody. This application further improves the sensitivity and stability of the chromatographic test strip for VEGF detection by modifying natural sporopollenin and sequentially coating its surface with a SiO2 shell and a hydroxyapatite nanocrystal mineral layer. This material utilizes its superior physicochemical stability, rich multi-level pore structure, and excellent biocompatibility and mechanical strength to further enhance the sensitivity and stability of the chromatographic test strip for VEGF detection.

Description

Technical Field

[0001] The present application relates to the field of biological detection, and more specifically, to a chromatography test paper for detecting VEGF in fingertip blood, and a preparation method and application thereof. Background Art

[0002] As a biomarker that plays a central role in tumor development and progression, as well as numerous pathological processes, vascular endothelial growth factor (VEGF) is invaluable for early disease detection, dynamic disease monitoring, and treatment effectiveness assessment. Accurate VEGF test results can provide physicians with a strong basis for developing personalized treatment plans, helping patients receive more timely and effective treatment. With the continuous advancement of biotechnology, the requirements for VEGF detection technology are becoming increasingly stringent, requiring not only guaranteed accuracy but also improved sensitivity and specificity to adapt to complex and changing clinical environments.

[0003] Currently, various types of microspheres, including fluorescent magnetic microspheres, quantum dot microspheres, and latex microspheres, are commonly used as carriers in immunochromatographic detection of VEGF. Fluorescent magnetic microspheres leverage their inherent magnetism to separate and enrich samples, facilitating detection. Quantum dot microspheres utilize their unique fluorescent properties to generate detection signals, resulting in high detection accuracy. Latex microspheres are widely used due to their relatively low cost. While these microspheres each have their advantages, they still suffer from low sensitivity when used in VEGF detection.

[0004] Existing carrier materials for immunochromatographic detection have significant drawbacks in terms of pigment loading, stability and nonspecific adsorption, which is the main reason for the low detection sensitivity. From the perspective of pigment loading, latex microspheres are difficult to carry a large amount of pigment, resulting in insufficient signal intensity during the detection process, affecting the ability to detect low-concentration VEGF molecules. In terms of stability, latex microspheres are stored in a high-temperature environment, and they are prone to deformation and rupture, resulting in pigment leakage. Although some solutions are coated with a SiO2 layer, it is still difficult to effectively avoid this problem. Due to the deformation of the latex microspheres, the SiO2 layer is easily damaged during the subsequent antibody coating process. In addition, these materials also have the problem of nonspecific adsorption. They are prone to unnecessary binding with other substances in the detection system, interfering with the normal output of the detection signal. This nonspecific binding can enhance the background signal, masking the actual detection signal, further reducing the sensitivity of the detection, and ultimately causing frequent false positive results, which greatly affects the accuracy and reliability of VEGF detection.

[0005] In view of this, this application is filed. Summary of the Invention

[0006] In order to solve the above technical problems, the present application provides a chromatography test paper for detecting VEGF in fingertip blood, and a preparation method and application thereof.

[0007] This application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a method for preparing a chromatographic test paper for detecting VEGF in fingertip blood, the chromatographic test paper comprising a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorption pad stacked in sequence, wherein the conjugation pad is coated with modified sporopollenin microspheres, and the surface of the modified sporopollenin microspheres is labeled with anti-VEGF monoclonal antibody and rabbit anti-mouse antibody;

[0009] The preparation method of the modified sporopollenin microspheres comprises:

[0010] (1) Sporopollenin is dispersed in a hydrophobic dye solution and vacuum-impregnated at 50-60°C for 5-6 hours, followed by centrifugation to obtain dye-loaded sporopollenin microspheres;

[0011] (2) The dye-loaded sporopollenin microspheres were mixed with TEOS solution, and ammonia was added and stirred at room temperature for 5-6 hours to obtain sporopollenin microspheres coated with SiO2 shell layer;

[0012] (3) The sporopollenin microspheres coated with SiO2 shell layer were immersed in a mineralization solution containing calcium chloride and potassium hydrogen phosphate, and incubated at 35-40 ° C for 8-12 hours to obtain modified sporopollenin microspheres with hydroxyapatite nanocrystals coated on the surface.

[0013] Furthermore, the method for labeling anti-VEGF monoclonal antibody or quality control antibody on the surface of the modified sporopollenin microspheres comprises:

[0014] dissolving the modified sporopollenin microspheres in Mes buffer solution, adding NHS / EDC for surface activation, and obtaining activated modified sporopollenin microspheres;

[0015] The activated modified sporopollenin microspheres were redispersed in PBS buffer solution, anti-VEGF monoclonal antibody or rabbit anti-mouse antibody was added, and the mixture was shaken and centrifuged. After removing the supernatant, BSA was added for blocking treatment and washing.

[0016] Furthermore, the mass ratio of the modified sporopollenin microspheres to the anti-VEGF monoclonal antibody or the rabbit anti-mouse antibody is 1:0.05-0.1; after adding the anti-VEGF monoclonal antibody or the rabbit anti-mouse antibody, the mixture is shaken and reacted at 2-6° C. for 10-14 hours.

[0017] Furthermore, after the BSA is added for blocking treatment, the above process further includes the step of passivating the modified sporopollenin microspheres:

[0018] The modified sporopollenin microspheres after BSA blocking treatment were dispersed in a 0.05-0.15 M polylysine-polyethylene glycol solution, shaken at 20-30° C. for 25-35 minutes, and washed by centrifugation.

[0019] Furthermore, in the above process of preparing the dye-loaded sporopollenin microspheres, the mass ratio of sporopollenin to the hydrophobic dye is 1 g:3-5 mg.

[0020] Furthermore, the mineralization liquid is obtained by dispersing calcium chloride dihydrate and potassium hydrogen phosphate in a mass ratio of (1-3):1 in a buffer solution with a pH of 7-8.

[0021] Furthermore, the coating method of the conjugate pad includes:

[0022] Dispersing modified sporopollenin microspheres labeled with anti-VEGF monoclonal antibody and modified sporopollenin microspheres labeled with rabbit anti-mouse antibody in a protective solution containing trehalose and PEG2000 to obtain a conjugate pad treatment solution;

[0023] The conjugate pad treating liquid is sprayed onto the glass fiber to obtain a conjugate pad.

[0024] Furthermore, a detection line and a quality control line are provided on the nitrocellulose membrane. The detection line is fixed with anti-VEGF antibodies for capturing the sporopollenin microsphere-VEGF complex; the quality control line is fixed with goat anti-rabbit antibodies for verifying the effectiveness of the detection process.

[0025] In a second aspect, the present application provides a chromatography test paper for detecting VEGF in fingertip blood, which is prepared by the above-mentioned preparation method.

[0026] In a third aspect, the present application provides a kit for detecting VEGF in fingertip blood, which includes the above-mentioned chromatography test paper.

[0027] In summary, this application has the following beneficial effects:

[0028] In order to solve the sensitivity and stability problems of VEGF detection, this application selects sporopollenin, a biopolymer derived from plant pollen, which has superb physical and chemical stability, rich multi-level pore structure, and excellent biocompatibility and mechanical strength. While being able to achieve a high loading of dye molecules, it is also sufficiently stable to avoid the deformation and breakage problems caused by long-term storage of traditional latex microspheres in high temperature and high humidity environments. At the same time, its internal natural multi-level pore structure can also play a molecular sieve role, intercepting matrix interfering substances such as large cell fragments and biomacromolecules, and pre-enriching small molecules of VEGF in the pores, increasing its local concentration, thereby improving the detection sensitivity to 15pg / ml.

[0029] This application modifies natural sporopollenin by sequentially coating its surface with a SiO2 shell (SiO2 layer) and a hydroxyapatite nanocrystal mineralized layer (HAP layer) to prevent leakage of adsorbed dye molecules and potential detection failure. The SiO2 layer, 20-50nm thick and mesoporous, helps intercept macromolecular interfering agents while providing active sites for coupling anti-VEGF monoclonal antibodies and quality control antibodies. The HAP layer, through hydroxyapatite nanocrystals, creates a seal around the SiO2 layer of the sporopollenin, enhancing its mechanical strength and further preventing dye leakage and the risk of SiO2 layer breakage.

[0030] In the preferred technical solution of the present application, in order to further avoid false positives and improve the accuracy of detection, after the modified sporopollenin particles are coupled with anti-VEGF monoclonal antibodies and quality control antibodies, the modified sporopollenin particles are passivated using polymerized lysine-polyethylene glycol to reduce nonspecific adsorption. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a linear fitting diagram in the performance test of this application. DETAILED DESCRIPTION

[0032] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0033] The technical solution of the present invention is:

[0034] The present application provides a method for preparing a chromatographic test paper for detecting VEGF in fingertip blood using modified sporopollenin microspheres, which comprises a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorption pad that are sequentially connected. Specifically:

[0035] (1) Sample pad: It is a polyester film, mainly used for loading and adsorbing samples.

[0036] (2) Binding pad: It is a glass fiber coated with two types of modified sporopollenin microspheres, namely modified sporopollenin microspheres labeled with anti-VEGF monoclonal antibody and modified sporopollenin microspheres labeled with rabbit anti-mouse antibody. Its function is to specifically bind to the target molecule VEGF in the sample and label it to facilitate subsequent retention and identification on the detection line.

[0037] The preparation method of modified sporopollenin microspheres includes:

[0038] a. Sporopollenin was dispersed in a hydrophobic dye solution and vacuum impregnated at 50-60°C for 5-6 hours, followed by centrifugation to obtain dye-loaded sporopollenin microspheres (SM).

[0039] Furthermore, the mass ratio of sporopollenin to hydrophobic dye is 1g:3-5mg, more preferably 1g:3.5-4.5mg, to ensure the optimal loading of the dye. Preferably, the vacuum impregnation temperature is 53-58°C and the impregnation time is 5.2-5.8h. The hydrophobic dye can be a common dye or a fluorescent dye commonly used in the biological field. Sudan red is used as an example in this application.

[0040] b. The dye-loaded sporopollenin microspheres were mixed with a tetraethoxysilane (TEOS) solution, and ammonia was added and stirred at room temperature for 5-6 hours to obtain sporopollenin microspheres coated with a SiO2 shell (abbreviated as SM@SiO2).

[0041] Furthermore, the mass volume ratio of sporopollenin microspheres to TEOS is 100 mg: 0.08-0.12 ml, and the reaction time is controlled at 5-6 h, so that the SiO2 shell is 30-50 nm and the surface has a mesoporous structure, avoiding the SiO2 shell being too thick and too dense.

[0042] c. Immerse the sporopollenin microspheres coated with a SiO2 shell in a mineralization solution containing calcium chloride and potassium hydrogen phosphate, and incubate with shaking at 35-40°C for 8-12 hours to obtain modified sporopollenin microspheres coated with hydroxyapatite nanocrystals (abbreviated as SM@SiO2-HAP).

[0043] By oscillating and incubating SM@SiO2 with the mineralizing solution, a mineralization reaction will occur on the surface of SM@SiO2 to form nano-needle-shaped crystals of hydroxyapatite (HAP), which are intertwined to form a dense network structure, which has a biomimetic effect and improves the overall mechanical strength of the material.

[0044] Furthermore, the mineralization solution is obtained by dispersing calcium chloride dihydrate (CaCl2·2H2O) and potassium hydrogen phosphate (K2HPO4) in a Tris-HCl buffer solution with a pH of 7-8 at a mass ratio of (1-3):1. Preferably, the mass-to-volume ratio of SM@SiO2 to the mineralization solution is (1.5-2.5) mg:1 ml, and the concentrations of CaCl2·2H2O and K2HPO4 in the mineralization solution are both 0.12-0.25 g / L.

[0045] (3) Nitrocellulose membrane: Located at one end of the conjugate pad, it is provided with a detection line and a quality control line. The detection line is fixed with anti-VEGF antibodies for capturing the sporopollenin microsphere-VEGF complex; the quality control line is fixed with goat anti-rabbit antibodies for verifying the effectiveness of the detection process.

[0046] (4) Absorption pad: mainly absorbs water and promotes chromatography.

[0047] (5) Preparation of chromatography test paper: Take a rubber plate and affix the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad to it in order to form a large plate. Then cut it into small strips 4 mm wide to obtain chromatography test paper. The chromatography test paper is packaged into a card cover and a sample loading hole is opened at the position corresponding to the sample pad.

[0048] The raw materials and sources used in this application are shown in Table 1:

[0049] Table 1. Raw materials and sources

[0050] Raw material components Manufacturer or CAS number effect Sporopollenin Shaanxi Mufan carrier Anti-VEGF monoclonal antibodies Sigma Binding to target molecules Anti-VEGF polyclonal antibody Sigma Intercept target molecules Rabbit anti-mouse antibody Absea Quality control antibodies Goat anti-rabbit antibody Absea Quality control antibodies Tetraethoxysilane TEOS 78-10-4 Silane agent Poly-lysine-polyethylene glycol Suzhou Beike passivating agent EDC / NHS Sigma Activator Nile Red Sigma dye nitrocellulose membrane Sartorius Conjugate pad

[0051] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0052] Preparation example of modified sporopollenin microspheres

[0053] Preparation Example 1

[0054] This preparation example provides a method for preparing modified sporopollenin microspheres, which comprises:

[0055] (1) Pretreatment of spore powder: After sorting the purchased spore powder, disperse the 20-30 μm spore powder in ethanol solution and perform ultrasonic treatment for 30 minutes;

[0056] (2) Pigment loading: Prepare a 5 mg / ml Nile red ethanol solution, disperse the pretreated sporopollenin powder (2 g) in 20 ml of Nile red ethanol solution, and perform vacuum impregnation at 55°C and -0.1 MPa for 5 h. Then, centrifuge at 8000 rpm for 10 min. Collect the microspheres and dry them to obtain Nile red loaded sporopollenin powder microspheres (SM). The dye loading amount was determined to be 0.35±0.05 μg dye / mg sporopollenin.

[0057] (3) Encapsulation of SiO2: 500 mg of sporopollenin microsphere powder was dispersed in 10 ml of ethanol, ultrasonically dispersed for 10 min, then added to 100 ml of ethanol, 3 ml of 28% ammonia water, and 0.5 ml of TEOS was added dropwise. After continuous stirring at room temperature for 6 h, the reaction was centrifuged and washed to obtain sporopollenin microspheres with a SiO2 shell layer on the surface (SM@SiO2). The thickness of the SiO2 shell was determined to be 40-50 nm.

[0058] (4) HAP mineralization: CaCl2·2H2O (0.245 g / L) and K2HPO4 (0.136 g / L) were dissolved in Tris-HCl buffer to obtain a mineralization solution; 100 mg of SM@SiO2 microspheres were immersed in 50 mL of the mineralization solution and shaken at 37°C and 120 rpm for 12 h. The microspheres were collected by filtration, washed with deionized water until neutral, and freeze-dried to obtain modified sporopollenin microspheres (SM@SiO2-HAP-1).

[0059] Preparation Example 2

[0060] The difference between this preparation example and Preparation Example 1 is that in the pigment loading step, a 3 mg / ml Nile red ethanol solution was prepared, and the pretreated spore powder (2 g) was dispersed in 20 ml of the Nile red ethanol solution, that is, the mass ratio of spore powder to Nile red was 1 g:3 mg. The resulting material was recorded as SM@SiO2-HAP-2.

[0061] Comparative Preparation Example 3

[0062] (5) The difference between this preparation example and Preparation Example 1 is that during the SiO2 encapsulation process, the volume of TEOS added was 0.8 mL, the stirring reaction time was 10 h, and the obtained material was recorded as SM@SiO2-HAP-3. The thickness of the SiO2 shell was measured to be 70-80 nm.

[0063] Comparative Preparation Example 4

[0064] The difference between this preparation example and Preparation Example 1 is that HAP mineralization treatment is not performed and the thickness of SiO2 is increased. Specifically: during the SiO2 encapsulation process, the volume of TEOS added is 0.8 mL, the stirring reaction time is 10 h, and after centrifugal washing and drying, the obtained material is recorded as SM@SiO2-4.

[0065] Example

[0066] Example 1

[0067] This embodiment provides a chromatography test paper for detecting VEGF in fingertip blood, and the preparation method thereof includes:

[0068] (1) Labeled antibodies:

[0069] 1 mg of modified sporopollenin microspheres (SM@SiO2-HAP-1) were dispersed in a Mes buffer solution at pH 6.0. After sonication for 5 minutes, 100 μl of EDC / NHS solution (both at a concentration of 5 mg / ml) was added dropwise and activated by vortexing at room temperature for 30 minutes. Subsequently, the solution was centrifuged at 8000 rpm for 10 minutes and redispersed in PBS buffer. After sonication for 3 minutes, 0.1 mg of anti-VEGF monoclonal antibody was added and the reaction was shaken at 4°C for 12 hours. Subsequently, 1% BSA was added for blocking for 1 hour and the solution was washed three times with PBS to obtain modified sporopollenin microspheres labeled with anti-VEGF monoclonal antibody.

[0070] Referring to the above method, modified sporopollenin microspheres labeled with rabbit anti-mouse antibodies were obtained.

[0071] (2) Preparation of conjugate pad:

[0072] Mix 100 μL of 25% trehalose aqueous solution, 100 μL of 10% PEG2000 aqueous solution and 125 μL of Tris-HCl buffer with a pH of 7.5, add 20 μL of modified sporopollenin microspheres labeled with anti-VEGF monoclonal antibody and 20 μL of modified sporopollenin microspheres labeled with anti-rabbit and anti-mouse antibodies, and add pure water to 1000 μL to obtain a conjugate pad treatment solution; spray the conjugate pad treatment solution onto the glass fiber and dry it at 40°C for 3 hours to obtain a conjugate pad.

[0073] (3) Preparation of nitrocellulose membrane:

[0074] Dilute the anti-VEGF polyclonal antibody to 0.4 mg / mL in PBS buffer, and dilute the goat anti-rabbit antibody to 0.8 mg / mL to prepare the working solutions. Streak the two working solutions onto a nitrocellulose membrane using a streak marker to create the test and control lines, respectively. The streak concentration is 1 μl / cm. The membrane is then dried at 50°C for 12 hours to obtain the nitrocellulose membrane.

[0075] (4) Preparation of test paper:

[0076] Take a plastic plate and affix a sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad to it in order to form a large plate. Then cut it into 4 mm wide strips to obtain chromatography test paper. The chromatography test paper is packaged in a card holder with a sample loading hole corresponding to the sample pad.

[0077] Example 2

[0078] The difference between this embodiment and embodiment 1 is that: in the process of labeling antibodies, the mass ratio of the modified sporopollenin microspheres to the anti-VEGF monoclonal antibody or the rabbit anti-mouse antibody is 1:0.05.

[0079] Example 3

[0080] The difference between this embodiment and embodiment 1 is that in the process of labeling the antibody, after adding BSA for blocking treatment, the modified sporopollenin microspheres are passivated.

[0081] A 0.1 mg / ml polylysine-g-PEG (PLL-g-PEG) PBS solution was prepared. The modified sporopollenin microspheres labeled with anti-VEGF monoclonal antibody or rabbit anti-mouse antibody were redispersed in the polylysine-g-PEG solution. The microspheres were shaken at room temperature for 30 minutes, centrifuged, and washed three times with PBS to remove free polymers to obtain the passivated labeled antibodies.

[0082] Example 4

[0083] The difference between this embodiment and embodiment 1 is that in the process of labeling the antibody, the modified sporopollenin microspheres provide SM@SiO2-HAP-2 for Preparation Example 2.

[0084] Comparative Example

[0085] Comparative Example 1

[0086] The difference between this comparative example and Example 1 is that in the process of labeling the antibody, the modified sporopollenin microspheres provide SM@SiO2-HAP-3 as in Preparation Example 3.

[0087] Comparative Example 2

[0088] The difference between this comparative example and Example 1 is that in the process of labeling the antibody, the modified sporopollenin microspheres provide SM@SiO2-4 for Preparation Example 4.

[0089] Comparative Example 3

[0090] This comparative example uses commercially available colored latex microspheres as carriers, and the method for labeling antibodies on their surfaces is shown in Example 1.

[0091] Performance testing

[0092] 1. Linear range

[0093] Using the chromatographic test paper provided in Example 1, a series of standard samples (VEGF) with concentrations ranging from 10 to 500 pg / ml were tested as follows:

[0094] Take the chromatography test paper provided in Example 1, add 10 μl of the standard sample to the sample loading well, wait for 3 minutes, then add 4 drops of loading buffer (containing 0.5 wt % Tween 80 and 0.1 wt % preservative), wait for 10 minutes, observe the color development with the naked eye, and measure the fluorescence peak using a fluorimeter.

[0095] Result judgment:

[0096] Positive: The test line and the quality control line show red at the same time;

[0097] Negative: the test line has no color and the quality control line shows red;

[0098] Invalid: The control line does not show color.

[0099] Each sample was measured three times in parallel, and the mean value (y) of the fluorescence peak was calculated. Then, a linear fit was performed with the standard sample concentration (x) as the independent variable, and the correlation coefficient (r) was calculated. The results are shown in Figure 2. Figure 1 As shown: the linear equation is: y = 0.0213x + 0.1792, and in the concentration range of 20pg / mL-160pg / mL, the linear correlation coefficient r is ≥ 0.9500.

[0100] 2. Clinical Sample Testing

[0101] The same method was used to test 5 clinical fingertip blood samples (after dilution), and the results were substituted into the above linear equation to obtain the VEGF content in the diluted clinical fingertip blood samples. The results are shown in Table 2:

[0102] Table 2. Clinical fingerstick blood dilution sample measurement results

[0103] Clinical samples VEGF content (pg / mL) Fingerstick blood sample 1 45.3 Fingerstick blood sample 2 37.8 Fingerstick blood sample 3 62.4 Fingerstick blood sample 4 48.5 Fingerstick blood sample 5 55.1

[0104] 3. Sensitivity measurement:

[0105] Using the same method, the test strips provided in Examples 1-4 and Comparative Examples 1-3 were used to test the standard samples with decreasing theoretical addition amounts, and the measurements were repeated three times. The average value was taken and the sensitivity was calculated. The results are shown in Table 3:

[0106] Table 3. Sensitivity test results

[0107] test strips Bond pad material Sensitivity (pg / mL) Example 1 <![CDATA[SM@SiO2-HAP-1]]> 15.6 Example 2 <![CDATA[SM@SiO2-HAP-1]]> 18.4 Example 3 <![CDATA[SM@SiO2-HAP-1]]> 16.4 Example 4 <![CDATA[SM@SiO2-HAP-2]]> 17.6 Comparative Example 1 <![CDATA[SM@SiO2-HAP-3]]> 34.1 Comparative Example 2 <![CDATA[SM@SiO2-4]]> 47.7 Comparative Example 3 Colored latex microspheres 84.6

[0108] As shown in Table 3, the test strips provided in Examples 1-4 of this application exhibited a VEGF sensitivity of 15-18 pg / mL, significantly higher than that of the colored latex microsphere test strips, demonstrating clinical significance. The sensitivity of Comparative Example 1 was 34.1 pg / mL, primarily due to the thick SiO2 coating on the surface, which hindered VEGF from entering the internal pores of the sporopollenin microspheres for pre-enrichment, resulting in low sensitivity. Comparative Example 2, lacking a HAP mineralized layer, also exhibited low sensitivity, potentially due to a small amount of dye leakage from the microspheres during centrifugation or damage to the SiO2 coating.

[0109] 4. Stability determination

[0110] The test strips provided in Examples 1-4 and Comparative Examples 1-3 were stored in a chamber at a temperature of 40° C. and a relative humidity of 75%. Samples were taken after 6 months and the sensitivity was calculated again using the above method. The results are shown in Table 4:

[0111] Table 4. Sensitivity test results

[0112] test strips Bond pad material Sensitivity (pg / mL) Example 1 <![CDATA[SM@SiO2-HAP-1]]> 18.2 Example 2 <![CDATA[SM@SiO2-HAP-1]]> 19.3 Example 3 <![CDATA[SM@SiO2-HAP-1]]> 15.1 Example 4 <![CDATA[SM@SiO2-HAP-2]]> 19.3 Comparative Example 1 <![CDATA[SM@SiO2-HAP-3]]> 38.7 Comparative Example 2 <![CDATA[SM@SiO2-4]]> 52.6 Comparative Example 3 Colored latex microspheres 122.4

[0113] It can be seen from Table 3 and Table 4 that after storage for 6 months under high humidity and high temperature environment, Examples 1-4 show better stability, and their detection sensitivity has only a slight increase. The sensitivity of the test strip of the colored latex microspheres of Comparative Example 3 has almost increased by 50%, and Comparative Example 1 and Comparative Example 2 have only a slight increase. This shows that the modified sporopollenin microspheres used in this application can improve the stability of the test strip, especially the storage stability under high temperature and high humidity environment.

[0114] 5. Anti-interference determination:

[0115] Triglycerides, bilirubin, and hemoglobin are common endogenous interfering substances. Therefore, the following three test samples were selected: a hyperlipidemic serum sample (triglycerides > 500 mg / dL), a hemolyzed sample (hemoglobin > 2 g / L), and a hyperbilirubinemic sample (total bilirubin > 20 mg / dL). A standard (VEGF) at a concentration of 50 pg / mL was added to each sample. The same method was used to determine the VEGF recovery rate using the test strips provided in Examples 1 and 3 and Comparative Example 3. The results are shown in Table 5:

[0116] Table 5. Anti-interference test results

[0117] Recovery rate Hyperlipidemic serum samples Hemolyzed samples High bilirubin sample Example 1 92.4% 95.1% 91.5% Example 3 98.7% 103.2% 96.28% Comparative Example 3 82.4% 86.1% 79.2%

[0118] As shown in Table 5, compared to Comparative Example 3, the test strips provided in Examples 1 and 3 of the present application showed VEGF recovery rates of 90-105% for the three test samples, indicating that the test strips are resistant to matrix interference. The best result was obtained in Example 3, indicating that passivation of the modified sporopollenin microspheres after antibody labeling helps reduce nonspecific adsorption and improves anti-interference ability.

[0119] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for preparing a chromatography test paper for detecting VEGF in fingertip blood, characterized in that: The chromatography test paper comprises a sample pad, a conjugation pad, a nitrocellulose membrane and an absorption pad which are sequentially overlapped, wherein the conjugation pad is coated with modified sporopollenin microspheres, and the surface of the modified sporopollenin microspheres is labeled with anti-VEGF monoclonal antibody and rabbit anti-mouse antibody; The preparation method of the modified sporopollenin microspheres comprises: (1) Sporopollenin is dispersed in a hydrophobic dye solution and vacuum-impregnated at 50-60°C for 5-6 hours, followed by centrifugation to obtain dye-loaded sporopollenin microspheres; (2) The dye-loaded sporopollenin microspheres were mixed with TEOS solution, and ammonia was added and stirred at room temperature for 5-6 hours to obtain sporopollenin microspheres coated with SiO2 shell layer; (3) The sporopollenin microspheres coated with SiO2 shell layer were immersed in a mineralization solution containing calcium chloride and potassium hydrogen phosphate, and incubated at 35-40 ° C for 8-12 hours to obtain modified sporopollenin microspheres with hydroxyapatite nanocrystals coated on the surface.

2. The method for preparing a chromatography test paper for detecting VEGF in fingertip blood according to claim 1, wherein The method for labeling anti-VEGF monoclonal antibody or rabbit anti-mouse antibody on the surface of modified sporopollenin microspheres comprises: Dispersing the modified sporopollenin microspheres in a Mes buffer solution, adding NHS / EDC for surface activation, and obtaining activated modified sporopollenin microspheres; The activated modified sporopollenin microspheres were redispersed in PBS buffer solution, anti-VEGF monoclonal antibody or rabbit anti-mouse antibody was added, and the mixture was shaken and centrifuged. After removing the supernatant, BSA was added for blocking treatment and washing.

3. The method for preparing a chromatography test paper for detecting fingertip blood VEGF according to claim 2, wherein: The mass ratio of the modified sporopollenin microspheres to the anti-VEGF monoclonal antibody or the rabbit anti-mouse antibody is 1:0.05-0.1; after adding the anti-VEGF monoclonal antibody or the rabbit anti-mouse antibody, the mixture is shaken and reacted at 2-6° C. for 10-14 hours.

4. The method for preparing a chromatography test paper for detecting fingertip blood VEGF according to claim 2, wherein After adding BSA for blocking treatment, the modified sporopollenin microspheres are passivated: The modified sporopollenin microspheres after BSA blocking treatment were dispersed in a 0.05-0.15 M polylysine-polyethylene glycol solution, shaken at 20-30° C. for 25-35 minutes, and washed by centrifugation.

5. The method for preparing a chromatography test paper for detecting VEGF in fingertip blood according to claim 1, wherein In the process of preparing the dye-loaded sporopollenin microspheres, the mass ratio of sporopollenin to the hydrophobic dye is 1 g:3-5 mg.

6. The method for preparing a chromatography test paper for detecting VEGF in fingertip blood according to claim 1, wherein: The mineralization liquid is obtained by dispersing calcium chloride dihydrate and potassium hydrogen phosphate in a buffer solution with a pH of 7-8 according to a mass ratio of (1-3):

1.

7. The method for preparing a chromatography test paper for detecting VEGF in fingertip blood according to claim 1, wherein: The coating method of the conjugate pad comprises: Dispersing modified sporopollenin microspheres labeled with anti-VEGF monoclonal antibody and modified sporopollenin microspheres labeled with rabbit anti-mouse antibody in a protective solution containing trehalose and PEG2000 to obtain a conjugate pad treatment solution; The conjugate pad treating liquid is sprayed onto the glass fiber to obtain a conjugate pad.

8. The method for preparing a chromatography test paper for detecting VEGF in fingertip blood according to claim 1, wherein: The nitrocellulose membrane is provided with a detection line and a quality control line. The detection line is fixed with anti-VEGF antibodies for capturing the sporopollenin microsphere-VEGF complex; the quality control line is fixed with goat anti-rabbit antibodies for verifying the effectiveness of the detection process.

9. A chromatography test paper for detecting VEGF in fingertip blood, characterized in that: The invention discloses a novel cellulose acetate copolymer comprising the steps of: preparing the cellulose acetate copolymer according to any one of claims 1 to 8; 10. A kit for detecting VEGF in fingertip blood, characterized in that: It comprises the chromatography test paper as claimed in claim 9.

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