CN115505067A - Selective targeting brain natriuretic peptide polymer and application thereof - Google Patents
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Abstract
The invention discloses a polymer capable of selectively targeting brain natriuretic peptide and application thereof. The invention designs and synthesizes a polymer capable of selectively targeting brain natriuretic peptide based on a bionic molecule design strategy by combining a calculation simulation model, a coarse graining model and a high-throughput screening technology, and researches the application of the polymer in the rapid detection of BNP. The polymer obtained by the invention has higher affinity and good selectivity, and can be applied to enrichment and detection of serum middle brain natriuretic peptide BNP instead of biological antibodies; the polymer is prepared by a chemical method, has higher stability, longer service life and stronger capability of resisting severe environment, and overcomes the defects of long preparation period, easy inactivation, high cost, immunogenicity and the like of the traditional biological antibody. The nano polymer is directly synthesized in a 96-well plate, and the quantitative detection of BNP in a serum sample can be realized within 15 min; the BNP can be visually detected by preparing the BNP into a fluorescent test strip.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a polymer capable of selectively targeting brain natriuretic peptide and application thereof.
Background
Heart failure refers to the condition of heart circulatory disturbance, which is caused by the failure of systolic and/or diastolic function of the heart, such as pulmonary congestion and vena cava congestion, due to the failure of adequate discharge of venous return blood from the heart. Heart failure is not an independent disease but the terminal stage of progression of heart disease. The B-type Brain Natriuretic Peptide (BNP) rapid diagnostic reagent can be used as an auxiliary diagnostic reagent for rapidly diagnosing heart failure and is increasingly applied.
In clinical work, serum BNP was less than 100pg/mL, which is the normal range. More than twice, it indicates heart failure. However, due to the complex components of the serum sample, the detection of the low-abundance BNP in the current real sample has the defects of high cost, high detection difficulty, long detection time and the like.
Disclosure of Invention
The invention aims to overcome the defects of high cost, low preparation efficiency, long screening period, difficult changeability and preservation, immunogenicity and the like in a detection technology taking a natural antibody and a monoclonal antibody as cores, designs and synthesizes a polymer capable of selectively targeting brain natriuretic peptide based on a bionic molecule design strategy and combined with a calculation simulation model, a coarse graining model and a high-throughput screening technology, and researches the application of the polymer in the rapid detection of BNP.
The preparation method of the polymer capable of selectively targeting the brain natriuretic peptide comprises the following steps: preparing a mixed reaction solution of N-isopropylacrylamide, N-tert-butylacrylamide, a charged functional monomer, fluorescent quantum dots, sodium dodecyl sulfate and a cross-linking agent, pouring the reaction solution into a 96-well plate, introducing nitrogen, adding an initiator solution, sealing, carrying out a polymerization reaction, removing the solution on the 96-well plate after the reaction is finished, and obtaining the polymer which is adhered to the bottom of the 96-well plate and can be used for selectively targeting the brain natriuretic peptide.
A preparation method of a fluorescent detection test strip for brain natriuretic peptide comprises the following steps: preparing a mixed reaction solution of N-isopropylacrylamide, N-tert-butylacrylamide, a charged functional monomer, fluorescent quantum dots, sodium dodecyl sulfate and a cross-linking agent, introducing nitrogen to discharge air, adding an initiator solution, sealing, carrying out polymerization reaction, dialyzing after the reaction is finished to obtain a polymer solution, mixing the polymer solution and fluorescent silica particles, pouring the mixture on a nitrocellulose membrane for suction filtration, and drying a filter membrane to obtain the fluorescent detection test strip for the brain natriuretic peptide.
The charged functional monomer is selected from one or more of acrylic acid, methacrylic acid, sodium vinylsulfonate, N- (3-aminopropyl) methacrylamide, (3-acrylamidopropyl) trimethyl ammonium chloride, 1-vinylimidazole, N- (3-dimethylaminopropyl) acrylamide, 4-vinylphenylboronic acid and N- (2-aminoethyl) acrylamide.
The molar ratio of the N-isopropyl acrylamide, the N-tertiary butyl acrylamide, the charged functional monomer and the cross-linking agent in the mixed reaction liquid is 30-70.
The cross-linking agent is N, N' -methylene bisacrylamide; the addition amount of the sodium dodecyl sulfate is 0.02-0.2mg/mL.
The polymerization reaction is free radical polymerization, the polymerization reaction temperature is 45-65 ℃, and the polymerization reaction time is 12-15h.
The initiator solution is ammonium persulfate solution or mixed solution of ammonium persulfate and tetramethyl ethylene diamine.
The fluorescent quantum dots are fluorescent carbon quantum dots, and the addition amount is 0.005-0.01mol/L.
The method for detecting the brain natriuretic peptide by the polymer capable of selectively targeting the brain natriuretic peptide comprises the following steps: and dropwise adding a serum sample on the polymer capable of selectively targeting the brain natriuretic peptide, measuring the fluorescence intensity value of the polymer at the position of 420nm by using an enzyme labeling instrument after reacting for 3-8min, and quantitatively calculating the BNP in the serum sample according to the measured fluorescence intensity value and a standard curve measured by using a sample with known concentration.
The use method of the fluorescent detection test strip for the brain natriuretic peptide comprises the following steps: and dripping the serum sample on a fluorescence detection test strip of the brain natriuretic peptide, and then placing the test strip under ultraviolet light to realize the detection of the brain natriuretic peptide according to the change of fluorescence color.
The invention has the following beneficial effects:
(1) The polymer obtained by the invention has higher affinity and good selectivity, and can be applied to enrichment and detection of serum middle brain natriuretic peptide BNP instead of biological antibodies; the polymer is prepared by a chemical method, has higher stability, longer service life and stronger capability of resisting severe environment, and overcomes the defects of long preparation period, easy inactivation, high cost, immunogenicity and the like of the traditional biological antibody.
(2) The invention combines the nanometer technology and the biomembrane interference technology to establish a high-flux screening system for the artificial antibody of the brain natriuretic peptide BNP. The method can obtain the affinity K between the nano polymer and the brain natriuretic peptide BNP within 20 minutes D And association and dissociation rates k on And k dis . Greatly shortens the screening time and is also suitable for the high-throughput screening of the artificial antibodies of other antigens.
(3) The nano polymer prepared by the method can be repeatedly used, and has the advantages of low cost, high accuracy, short detection time, strong specificity and selectivity, high reliability and the like; and the synthesis process and the regeneration process are simple, and the method is suitable for detection application of various brain natriuretic peptide BNP. The BNP can be visually detected by preparing the BNP into a fluorescent test strip; the polymer embedding the quantum dots is directly synthesized in a 96-well plate, and quantitative detection of BNP in a serum sample can be realized within 15min through fluorescence detection.
Drawings
FIG. 1 is a scanning electron micrograph of polymers of different particle sizes prepared in example 1.
FIG. 2 is a real-time binding dissociation curve and its fit between different concentrations of polymer prepared in example 1 and BNP.
FIG. 3 is a graph comparing the fluorescence intensity of pure water and blue fluorescent quantum dot solution under the irradiation of an ultraviolet lamp.
Fig. 4 is a transmission electron microscope image of blue fluorescent quantum dots.
FIG. 5 is a graph showing the comparison of fluorescence intensity of pure water and a red fluorescent quantum dot solution under irradiation of an ultraviolet lamp.
Fig. 6 is a transmission electron microscope image of red fluorescent quantum dots.
FIG. 7 is a scanning electron microscope photograph of fluorescent silica particles in example 2.
Fig. 8 is a scanning electron microscope of the test strip for fluorescence detection of brain natriuretic peptide prepared in example 2.
FIG. 9 is the result of the visual detection of BNP at different concentrations in a serum sample by the fluorescent detection test strip for brain natriuretic peptide of example 2.
FIG. 10 shows the results of 10-fold repeated measurements of different batches using serum samples of the same concentration (BNP concentration of 150 pg/mL) in example 1.
FIG. 11 shows the results of 10-fold repeated measurements of the same serum sample (BNP concentration of 150 pg/mL) in application example 1.
Detailed Description
The design strategy of the polymer is to utilize a bionic molecule design method to research the interaction of BNP-flexible polymer by simulating the protein-protein interaction. The protein realizes the specific recognition of a specific binding domain of the protein through various interactions such as hydrogen bonds, electrostatic force, hydrophobicity and the like, so that amino acid-like functional monomers with positive electricity, negative electricity, hydrophobicity and easiness in forming the hydrogen bonds are selected to construct a monomer combined chemical library, a coarse graining simulation method is adopted to screen a monomer formula, the sequence structure and the spatial distribution of a polymer are regulated and controlled by adjusting the proportion of the functional monomers, the monomer formula is further obtained by a high-throughput screening method, and the polymer capable of selectively targeting the human brain natriuretic peptide BNP is prepared by inverse emulsion polymerization or precipitation polymerization.
Synthesis of blue fluorescent carbon quantum dots:
weighing 4.2g of citric acid solid in a 50mL centrifuge tube, adding 40mL of water to completely dissolve the solid, adding 1.34mL of ethylenediamine solution (the molar ratio of citric acid to ethylenediamine is 1:1), stirring to uniformly disperse the solution, transferring the liquid to a polytetrafluoroethylene reaction kettle, and placing the reaction kettle in an oven at 200 ℃ for reaction for 5 hours; after the reaction is finished, cooling to room temperature, dialyzing for 1 day by using a dialysis membrane with the molecular weight cutoff of 1000Da, and collecting for later use after the dialysis is finished. The synthesized fluorescent carbon quantum dots appeared blue (fig. 3), and the transmission electron microscope thereof is shown in fig. 4.
Synthesis of red fluorescent carbon quantum dots:
adding 0.1g of p-phenylenediamine and 1mL of ammonia water into 10mL of ethanol solution, ultrasonically stirring for 10min to dissolve the p-phenylenediamine and the ammonia water, then transferring the obtained solution into a polytetrafluoroethylene reaction kettle, placing the polytetrafluoroethylene reaction kettle into an oven, reacting for 6h at 180 ℃, naturally cooling to room temperature after the reaction is finished, diluting the obtained carbon quantum dots with ethanol to obtain red fluorescent quantum dots (figure 5), wherein a transmission electron microscope of the red fluorescent quantum dots is shown in figure 6.
Example 1: preparation of brain natriuretic peptide BNP artificial antibody based on change of nano polymer functional monomer composition ratio
1. Design and synthesis of nano-polymers
Preparing a mixed solution of the components with the total molar concentration of 65mM in a 96-well plate, wherein the molar percentage of N-isopropylacrylamide is 53mol%, the molar percentage of the charged functional monomer is shown in the table 1, the molar percentage of N, N' -methylenebisacrylamide is 2mol%, and the balance is N-tert-butylacrylamide; and continuously adding blue fluorescent carbon quantum dots and sodium dodecyl sulfate, wherein the addition amount of the blue fluorescent carbon quantum dots is 0.01mol/L, the addition amount of the sodium dodecyl sulfate is 0.2mg/mL, introducing nitrogen, then adding an ammonium persulfate solution, wherein the addition amount of the ammonium persulfate is 0.0035mol/L, sealing the pore plate by using a sealing film, carrying out polymerization reaction at 65 ℃ for 12 hours, then taking out the supernatant solution in the 96 pore plate, and adhering the supernatant solution to the bottom of the pore plate to obtain the polymer. By changing the composition ratio of the basic monomer and the functional monomer, the synthesized polymers are different, and a certain support is provided for rapidly screening the nano polymer with high affinity to BNP by utilizing the high efficiency of synthesis in the pore plate.
2. Screening of polymers
Selecting BNP as target substance, using polymer as bionic antibody, and utilizing organismThe affinity between the polymer and the brain natriuretic peptide BNP is measured by a membrane interference technique (BLI), and the biosensor is used for fixing the brain natriuretic peptide BNP in an interaction molecule to form a biological membrane layer. The nanoparticles to be tested are then placed in a detection cell and the thickness of the biolayer increases when the interaction of the polymer and BNP occurs. The binding dissociation time is used as an abscissa, the increment of the signal intensity caused by the drift of the interference curve is used as an ordinate, a standard curve is drawn, and the binding affinity K between the nanoparticles and the BNP is fitted D And association and dissociation rates k on And k dis 。
Determination of affinity constant K of polymer composed of different monomer proportions to adsorption of BNP by biological film interference technique (BLI) D The values, results are shown in figure 2 and table 1, indicating that a specific ratio of polymers has a very high affinity and selectivity for BNP, comparable to natural antibodies.
TABLE 1 affinity constant K of adsorption of BNP respectively for polymers prepared from different monomer proportions D Value of
According to the test results in table 1, the polymer prepared from acrylic acid with 6.5mol% of functional monomer has good affinity to BNP, and the polymers used in example 2 were synthesized using the monomer ratio and subjected to subsequent studies.
Example 2:
synthesis of fluorescent silica: mixing 25.5mL of anhydrous ethanol and 4.5mL of deionized water, transferring the mixture into a 100mL flask, stirring the mixture at the room temperature at the rotation speed of 620r/min by using magnetic stirring, simultaneously adding 900 mu L of tetraethyl orthosilicate (TEOS) solution, stirring the mixture for 10min, adding 12mL of dialyzed red fluorescent carbon quantum dot solution into the flask, continuing to stir the mixture for 40min, and finally continuing to add 1.2mL of ammonia water into the reaction system for continuous stirringAnd reacting for 6h. After the reaction is finished, centrifuging the obtained solution at the rotating speed of 10000r/min for 8min and discarding supernatant, dissolving the obtained precipitate with ethanol, centrifuging at the rotating speed of 10000r/min for 8min and discarding supernatant (repeating for 2 times), dissolving the obtained precipitate with deionized water, centrifuging at the rotating speed of 10000r/min for 8min and discarding supernatant (repeating for 2 times), adding water (totally 12 ml) into the finally obtained precipitate, and uniformly dispersing to obtain fluorescent silicon dioxide (SiO) 2 @ R), as shown in fig. 7.
Preparing a brain natriuretic peptide fluorescence detection test strip:
preparing 50ml of mixed solution with the total molar concentration of all components being 65mM, wherein the molar percentage content of N-isopropylacrylamide is 53mol%, the molar percentage content of N-tert-butylacrylamide is 38.5mol%, the molar percentage content of acrylic acid is 6.5mol%, and the molar percentage content of N, N' -methylenebisacrylamide is 2mol%; continuously adding blue fluorescent carbon quantum dots and sodium dodecyl sulfate, wherein the addition amount of the blue fluorescent carbon quantum dots is 0.01mol/L, and the addition amount of the sodium dodecyl sulfate is 0.2mg/mL; introducing nitrogen to discharge air, adding 1ml of initiator solution with the concentration of 0.175mol/L, sealing, carrying out polymerization reaction, dialyzing after the reaction is finished to obtain polymer solution, mixing the polymer solution and the fluorescent silica particle solution according to the volume ratio of 3:1, pouring the mixture on a nitrocellulose membrane for suction filtration, drying the filter membrane, and shearing the filter membrane into a fluorescent test strip of 1cm x 1cm. (A scanning electron microscope of the test strip for fluorescence detection of brain natriuretic peptide is shown in FIG. 8)
And then, placing the obtained fluorescent test strip at a fixed position on a table, adjusting and fixing the position of the portable ultraviolet analyzer, and keeping the positions of the ultraviolet analyzer and the fluorescent test strip fixed. And then, firstly, recording the fluorescence color of the test strip before the sample is dripped under the irradiation of an ultraviolet lamp with the wavelength of 365nm, then dripping a serum sample or a standard product containing BNP with different concentrations, recording the fluorescence color of the test strip after the sample is dripped again after 5min, and finally comparing the change of the fluorescence color of the test strip before and after the sample is dripped and further analyzing the change.
BNP in a serum sample can be combined with the polymer after contacting the polymer, so that the fluorescence intensity of fluorescent quantum dots in the polymer changes and even quenches, and visual detection of a sample to be detected can be realized by collecting the change condition of fluorescence of the test strip before and after dripping the sample by using a mobile phone APP. (the detection results are shown in FIG. 9) application example 1: quantitative detection of serum sample BNP by utilizing enzyme-linked immunosorbent assay to measure change of fluorescence intensity of polymer
Detecting a polymer synthesized at the bottom of a pore plate by using acrylic acid with 6.5mol% of functional monomer in the embodiment 1, measuring the fluorescence intensity value of the polymer at 420nm by using a microplate reader, preparing a series of differences of fluorescence intensities before and after serum measurement reaction with known BNP concentration, and drawing a calibration curve; and then, dripping 20 mu L of a serum sample to be detected into the pore plate, measuring the fluorescence intensity value of the sample in the pore plate at the position of 420nm after reaction by using the enzyme labeling instrument again after the reaction is carried out for 5min, and carrying out quantitative detection on BNP in the serum sample according to the change of the fluorescence intensity value and the measured standard curve.
The feasibility of the method is verified, and the relative standard deviation RSD of 10 repeated determinations of serum samples with the same concentration and different batches is 7.3 percent; the relative standard deviation RSD of the same serum sample was 2.33% after 10 replicates. Table 2 shows the measurement values of 10 replicates of different batches of the same serum sample concentration (BNP 150 pg/mL). Table 3 shows the measurement values obtained by repeating the measurement 10 times on the same serum sample. Table 4 shows the actual values and measured values of different serum samples.
TABLE 2
TABLE 3
| Sample number | BNP concentration (pg/mL) | Measured concentration (pg/mL) |
| 1 | 150 | 161.89 |
| 2 | 150 | 168.36 |
| 3 | 150 | 159.48 |
| 4 | 150 | 161.90 |
| 5 | 150 | 165.54 |
| 6 | 150 | 163.34 |
| 7 | 150 | 165.81 |
| 8 | 150 | 167.34 |
| 9 | 150 | 155.63 |
| 10 | 150 | 163.65 |
TABLE 4
| Sample number | BNP concentration (pg/mL) | Measured concentration (pg/mL) |
| 1 | 120 | 128.66 |
| 2 | 150 | 156.59 |
| 3 | 250 | 226.04 |
The present invention is not limited to the above-described examples, and various changes can be made without departing from the spirit and scope of the present invention within the knowledge of those skilled in the art.
Claims (10)
1. A preparation method of a polymer capable of selectively targeting brain natriuretic peptide is characterized by comprising the following steps: preparing a mixed reaction solution of N-isopropylacrylamide, N-tert-butylacrylamide, a charged functional monomer, fluorescent quantum dots, sodium dodecyl sulfate and a cross-linking agent, pouring the reaction solution into a 96-well plate, introducing nitrogen, adding an initiator solution, sealing, carrying out a polymerization reaction, removing the solution on the 96-well plate after the reaction is finished, and obtaining the polymer which is adhered to the bottom of the 96-well plate and can be used for selectively targeting the brain natriuretic peptide.
2. A preparation method of a brain natriuretic peptide fluorescence detection test strip is characterized by comprising the following steps: preparing a mixed reaction solution of N-isopropylacrylamide, N-tert-butylacrylamide, a charged functional monomer, fluorescent quantum dots, sodium dodecyl sulfate and a cross-linking agent, introducing nitrogen to discharge air, adding an initiator solution, sealing, carrying out polymerization reaction, dialyzing after the reaction is finished to obtain a polymer solution, mixing the polymer solution and fluorescent silica particles, pouring the mixture on a nitrocellulose membrane, carrying out suction filtration, and drying the filter membrane to obtain the fluorescent detection test strip for the brain natriuretic peptide.
3. The method according to claim 1 or 2, wherein the charged functional monomer is selected from one or more of acrylic acid, methacrylic acid, sodium vinylsulfonate, N- (3-aminopropyl) methacrylamide, (3-acrylamidopropyl) trimethylammonium chloride, 1-vinylimidazole, N- (3-dimethylaminopropyl) acrylamide, 4-vinylphenylboronic acid, and N- (2-aminoethyl) acrylamide.
4. The preparation method according to claim 1 or 2, wherein the molar ratio of N-isopropylacrylamide, N-tert-butylacrylamide, the charged functional monomer, and the crosslinking agent in the mixed reaction solution is in the range of 30 to 70.
5. The process according to claim 1 or 2, wherein the crosslinking agent is N, N' -methylenebisacrylamide; the addition amount of the sodium dodecyl sulfate is 0.02-0.2mg/mL.
6. The method according to claim 1 or 2, wherein the polymerization is a free radical polymerization, the polymerization temperature is 45-65 ℃ and the polymerization time is 12-15 hours.
7. The preparation method according to claim 1 or 2, characterized in that the initiator solution is ammonium persulfate solution or a mixed solution of ammonium persulfate and tetramethylethylenediamine.
8. The preparation method according to claim 1 or 2, wherein the fluorescent quantum dots are fluorescent carbon quantum dots, and the addition amount is 0.005-0.01mol/L.
9. The method for detecting the brain natriuretic peptide through the polymer capable of selectively targeting the brain natriuretic peptide prepared by the method of claim 1, wherein the method for detecting the brain natriuretic peptide is as follows: and dropwise adding a serum sample on the polymer capable of selectively targeting the brain natriuretic peptide, measuring the fluorescence intensity value of the polymer at the position of 420nm by using an enzyme labeling instrument after reacting for 3-8min, and quantitatively calculating the BNP in the serum sample according to the measured fluorescence intensity value and a standard curve measured by using a sample with known concentration.
10. The use method of the fluorescence detection test strip for brain natriuretic peptide prepared by the method of claim 2 is characterized in that the use method comprises the following steps: and dripping the serum sample on a fluorescence detection test strip of the brain natriuretic peptide, and then placing the test strip under ultraviolet light to realize the detection of the brain natriuretic peptide according to the change of fluorescence color.
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