CN110577616B - Preparation method and application of non-biological nano artificial antibody of specific targeting Alzheimer's disease marker - Google Patents
Preparation method and application of non-biological nano artificial antibody of specific targeting Alzheimer's disease marker Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a non-biological nano artificial antibody of a specific targeting Alzheimer disease marker. Testing the capability of the synthesized nano artificial antibody for effectively capturing the pathological marker of the Alzheimer's disease by utilizing a biomembrane interference technology, wherein the affinity constant K of the nano artificial antibody to the markerDThe value can reach 8.26 multiplied by 10‑10M is equivalent to an antibody and has excellent selectivity, high-selectivity enrichment of the low-abundance Alzheimer disease pathological marker in a serum sample can be realized by combining magnetic nanoparticles, an integral column or a microfluidic chip, and high-sensitivity detection of the Alzheimer disease pathological marker can be realized by methods such as high-sensitivity Raman spectroscopy, fluorescent quantitative detection, an ELISA kit, a chemiluminescence kit and the like.
Description
Technical Field
The invention belongs to the field of biological nano-medicine, and particularly relates to a preparation method and application of a non-biological nano artificial antibody of a specific targeting Alzheimer's disease marker.
Background
Alzheimer's Disease (AD) is a progressive neurodegenerative disease, an incurable, progressive, fatal degenerative nervous system disease. This disease causes a disorder in the ability of a patient to listen, speak, read and write, and fails to perform the life of a normal person, thus increasing the burden of family life. The number of AD patients will increase sharply with the population of the middle and old aged people reaching the peak, and the number of AD patients in China will reach 893 ten thousands by 2020, which will bring a heavy burden to the society. Amyloid cerebrovascular disease (CAA), senile plaque and neurofibrillary tangle are recognized as three pathological features of Alzheimer's disease, but the exact pathogenesis of the disease is not clear, and no effective treatment method or medicament can reverse and cure AD.
The most typical histopathological features of AD are plaques formed by amyloid beta in the central nervous system, i.e., abnormal deposition of amyloid beta, and extracellular abnormally aggregated amyloid beta acting on glial cells and neurons through various cellular cascades such as inflammatory reaction and free radical reaction, which finally cause abnormal and even death of neuron function and further cause cognitive impairment. Another hallmark pathological feature is the abnormal aggregation of hyperphosphorylated tau protein in nerve cells to form neurofibrillary tangles, which can block the neuronal trophic transport channels, eventually leading to the hypofunction of neurons until death and atrophy.
Natural antibodies are widely used for selective recognition and detection of AD markers. However, the natural antibody is mainly obtained by animal immunization, and has the defects of high cost, low preparation efficiency, long screening period, difficult changeability and preservation, immunogenicity and the like, and the monoclonal antibody also has the problem of large batch performance difference and has great limitation in practical application.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a preparation method and application of a non-biological nano artificial antibody specifically targeting an Alzheimer's disease marker, and the three-dimensional structure of a polymer nanoparticle is adjusted to create a nanoparticle-based artificial antibody with high protein-like specificity and reversible binding property.
The technical scheme is as follows:
a preparation method of a non-biological nano artificial antibody specifically targeting an Alzheimer's disease marker comprises the following steps: n-isopropyl acrylamide, N-tert-butyl acrylamide, sodium dodecyl sulfate, a charged functional monomer, N, N' -methylene bisacrylamide serving as a cross-linking agent, an Alzheimer disease pathological marker serving as a template molecule, and a non-biological nano artificial antibody obtained by eluting the template molecule after polymerization in a water phase under the action of an initiator.
Preferably, the pathological marker of alzheimer's disease comprises beta-amyloid, Tau protein, whole protein of hyperphosphorylated protein, or stable polypeptide fragment thereof.
Preferably, the charged functional monomer comprises any one or more of N- (3-aminopropyl) methacrylic acid, acrylic acid, methacrylic acid, 1-vinylimidazole, N- (3-dimethylaminopropyl) methacrylamide, acrylamide, (3-acrylamidopropyl) trimethylammonium chloride or N- (2-aminoethyl) acrylamide.
Preferably, N-isopropylacrylamide is used in an amount of 5 to 60 wt.%, N-tert-butylacrylamide is used in an amount of 5 to 50 wt.%, charged functional monomer is used in an amount of 0.5 to 20 wt.%, and N, N' -methylenebisacrylamide is used in an amount of 0.5 to 10 wt.%.
Preferably, the polymerization method is any one of inverse emulsion polymerization, precipitation polymerization, or radical polymerization.
Preferably, the initiator is ammonium persulfate or azobisisobutyronitrile.
Preferably, the polymerization reaction temperature under the nitrogen atmosphere is 20-70 ℃, and the polymerization reaction time is 3-36 h.
Preferably, the template molecule elution solution is any one of NaCl, sodium citrate, glycine or sodium dodecyl sulfate, or the elution of the template molecule is realized by changing the temperature and pH value.
Preferably, the particle size of the nanoparticle artificial antibody is 10-3000 nm.
The non-biological nano artificial antibody of the specific targeting Alzheimer disease marker prepared by the method can be used for combining magnetic nanoparticles, monolithic columns or microfluidic chips to realize high selective enrichment of low-abundance Alzheimer disease pathological markers in serum samples. The obtained nano artificial antibody combined with gold nanoparticles, quantum dots, fluorescent reagent or therapeutic drugs can be used for targeted therapy of Alzheimer's disease or targeted molecular imaging and diagnosis of senile plaque and neuron fiber knot winding, and can realize high-sensitivity detection of pathologic markers of Alzheimer's disease by methods such as high-sensitivity Raman spectroscopy, fluorescent quantitative detection, ELISA (enzyme-linked immunosorbent assay) kit, chemiluminescence kit, test paper strip, electrochemical sensor, immunoturbidity, immunochromatography, ultrasonic detection, CT (computed tomography) detection, nuclear magnetic detection and the like.
The invention utilizes biomembrane interference technology (BLI) to determine the affinity between the non-biological nanoparticle artificial antibody and the pathological marker of the Alzheimer's disease, and the biosensor fixes the pathological marker of the Alzheimer's disease. Then placing the nano particles to be detected in a detection pool, and increasing the thickness of a biological layer when the artificial antibody and the marker interact; 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 artificial antibody of the non-biological nano-particles and the marker is fittedDAnd association and dissociation rate constants konAnd koff。
The technical effects obtained by the invention are as follows:
(1) the non-biological nano artificial antibody targeting the pathological marker of the Alzheimer's disease has higher selectivity, and can replace a biological antibody to be applied to the detection of the pathological marker of the Alzheimer's disease in a brain tissue slice; the artificial antibody is prepared by a chemical method, has high stability, long service life and strong capability of resisting severe environment, and overcomes the defects of long preparation period, easy inactivation, high cost 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 pathological marker of the Alzheimer's disease. The method can obtain the affinity K between the nano particles and the marker within 20 minutesDAnd association and dissociation rates konAnd koffGreatly shortens the screening time and is also suitable for the high-throughput screening of artificial antibodies of other antigens.
(3) The artificial antibody prepared by the method can be repeatedly used, and the cost is greatly reduced; and the synthesis process and the regeneration process are simple, and the method is suitable for the diagnosis and treatment application of the pathological marker of the Alzheimer's disease in the brain tissue slice.
(4) The artificial antibody prepared by the method has wide application, and can realize high-selectivity enrichment of low-abundance Alzheimer disease pathological markers in serum samples by combining magnetic nanoparticles, monolithic columns or microfluidic chips. The obtained nano artificial antibody combined with gold nanoparticles, quantum dots, fluorescent reagent or therapeutic drugs can be used for targeted therapy of Alzheimer's disease or targeted molecular imaging and diagnosis of senile plaque and neuron fiber knot winding, and can realize high-sensitivity detection of pathologic markers of Alzheimer's disease by methods such as high-sensitivity Raman spectroscopy, fluorescent quantitative detection, ELISA (enzyme-linked immunosorbent assay) kit, chemiluminescence kit, test paper strip, electrochemical sensor, immunoturbidity, immunochromatography, ultrasonic detection, CT (computed tomography) detection, nuclear magnetic detection and the like.
Drawings
FIG. 1 is a scanning electron microscope image of nanoparticle artificial antibodies of different particle sizes.
Fig. 2 is a real-time binding dissociation curve and its fit line between different concentrations of artificial antibodies and pathological markers of alzheimer's disease.
FIG. 3 is the transmission electron microscope image and the scanning electron microscope image of the non-biological nano artificial antibody coated nano gold ball.
Fig. 4 is a surface enhanced raman spectrum of the nano artificial antibody raman probe.
Fig. 5 is raman imaging of nanoparticle artificial antibodies after binding to pathological markers of alzheimer's disease on brain tissue sections.
Detailed Description
In order to make the technical scheme of the present invention better understood by those skilled in the art, the following describes in detail the preparation method and use of a non-biological nano artificial antibody specifically targeting the alzheimer's disease marker provided by the present invention with reference to the examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.
The first embodiment is as follows: preparation of non-biological nano artificial antibody targeting Alzheimer disease pathological marker based on nano particle three-dimensional structure modified reconstruction recognition region
1. Design and synthesis of nanoparticles
N-isopropylacrylamide (58-X mol%), the charged monomer N- (3-dimethylaminopropyl) methacrylamide (X mol%), N-tert-butylacrylamide (40 mol%), the crosslinker N, N' -methylenebisacrylamide (2 mol%) and sodium dodecylsulfonate (10mg) were dissolved in water to give a total monomer concentration of 130 mM. After the initiator was added, polymerization was carried out at 65 ℃ for 3 hours under a nitrogen atmosphere with a magnetic stirrer. The polymerized solution was purified by dialysis with an excess of pure water, and the polymer nanoparticles were obtained after freeze-drying (FIG. 1).
2. Preliminary screening of Artificial antibodies
The selected target protein is beta-amyloid, but the complete beta-amyloid is expensive and not easy to obtain, wherein a stable polypeptide fragment of the A beta protein is selected as a target object, molecularly imprinted polymer nanoparticles are used as bionic antibodies, the affinity between the nanoparticle artificial antibodies and the beta-amyloid is measured by using a biomembrane interference technology (BLI), and the biosensor is used for fixing the beta-amyloid in interacting molecules to form a biomembrane layer. Then the nano-particles to be detected are placed in a detection pool, and when the interaction of the artificial antibody and the beta-amyloid protein occurs, the thickness of the biological layer is increased. 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 beta-amyloid protein is fittedDAnd association and dissociation rates konAnd koff。
3. Molecular imprinting technology for improving affinity and selectivity of artificial antibody
A polypeptide fragment which has high K with beta-amyloid protein is preliminarily screenedDAfter the nanoparticles are added, in order to further improve the affinity and selectivity of the nanoparticles to beta-amyloid, a molecular imprinting technology is adopted to improve humanSpecificity and selectivity of the antibody. The synthesis of imprinted polymers (MIPs) is as follows: n-isopropylacrylamide (25 mol%), N- (3-dimethylaminopropyl) methacrylamide (33 mol%), N-tert-butylacrylamide (40 mol%), N, N' -methylenebisacrylamide (2 mol%), sodium dodecylsulfate (10mg) and 10mg of beta-amyloid polypeptide fragments were dissolved in water to give a total monomer concentration of 130 mM. After adding an initiator ammonium persulfate, a polymerization reaction was carried out at 45 ℃ for 12 hours under a nitrogen atmosphere with a magnetic stirrer. And then adding 0.04mol of NaCl, continuously stirring for 30 minutes at room temperature to elute the template polypeptide, finally purifying the polymerized solution by dialysis with excessive pure water (changing water three times a day), and freeze-drying to obtain the molecularly imprinted polymer nanoparticles. Similarly, K between MIP and beta-amyloid obtained by BLI assayDValue and K between the two hetero-proteins (apolipoprotein E, heme oxygenase, plasma gelsolin, amyloid precursor protein)DThe value is obtained. Non-imprinted polymers (NIPs) synthesized without the addition of short beta-amyloid peptide fragments were also tested in the same manner (FIG. 2).
Example two: raman imaging of beta-amyloid nano artificial antibody on brain tissue slice
First, a gold nanosphere with a smaller diameter was synthesized by mixing 0.0255g of HAuCl4Dissolving in 255g of water, heating to boil, adding 15g of sodium citrate aqueous solution with the mass fraction of 1% into the solution, continuing to boil for 15min after the solution is in wine red, and naturally cooling to room temperature. And then the nano gold ball is concentrated by 20 times through centrifugation. Connecting the concentrated nanogold spheres with a DTNB dye, adding the nanogold spheres connected with the dye in the process of synthesizing the nanoparticles, wrapping the nanogold spheres in the nanoparticles, and using a Transmission Electron Microscope (TEM) and a Scanning Electron Microscope (SEM) as shown in figure 3. Raman depth scan was performed to determine the characteristic peaks (fig. 4).
And then, carrying out xylene and ethanol treatment with gradient concentration on the paraffin-encapsulated human brain tissue section, dropwise adding the synthesized nano particles onto the section, placing the section in a wet box, incubating for 60min at room temperature, washing the section with PBS (phosphate buffer solution) and NaCl solution with the concentration of 0.1M, drying the section with nitrogen, and carrying out Raman imaging.
Example three: beta-amyloid nano artificial antibody for fluorescence imaging on brain tissue section
First, after the synthesized nanoparticles were dialyzed and activated, 5mg of EDC and 2.5mg of NHS were added to 1mL of the nanoparticle solution and activated for 12h in a shaker at 4 ℃. Then 10. mu.L of FITC was added to each mL of nanoparticle suspension, and the mixture was reacted for 24 hours in a shaker at 4 ℃ and protected from light.
And then, carrying out xylene and ethanol treatment with gradient concentration on the paraffin-encapsulated human brain tissue section, dropwise adding the synthesized nano particles onto the section, placing the section in a wet box, incubating for 60min at room temperature, washing the section with PBS (phosphate buffer solution) and NaCl solution with the concentration of 0.1M, and drying by using nitrogen. Then laser confocal imaging is carried out.
Example four: beta-amyloid nano artificial antibody drug-loaded elimination of senile plaques on brain tissue slices
Beta-cyclodextrin can regulate the peptide aggregation process of beta-amyloid protein and reduce the fibrillation and the neurotoxicity of the beta-cyclodextrin.
First, after the synthesized nanoparticles were dialyzed and activated, 5mg of EDC and 2.5mg of NHS were added to 1mL of the nanoparticle solution and activated for 12h in a shaker at 4 ℃. Dissolving beta-cyclodextrin in a buffer solution with the pH value of 7.4, adding the beta-cyclodextrin into the nanoparticle solution according to the mass ratio of 10:1, reacting for 24 hours in a shaking table at the temperature of 4 ℃, and centrifuging to remove a supernatant after the reaction is finished.
And then, treating the paraffin-encapsulated human brain tissue section with dimethylbenzene and ethanol with gradient concentration, dropwise adding activated nanoparticles onto the section, placing the section in a wet box, incubating for 120min at room temperature, washing the section with PBS (phosphate buffer solution) and NaCl solution with the concentration of 0.1M, drying the section with nitrogen, performing Raman imaging (figure 5), and observing the reduction condition of the senile plaques.
The invention discloses a preparation method and application of a non-biological nano artificial antibody of a specific targeting Alzheimer's disease marker, which are implemented byThe gel nanoparticle artificial antibody is formed by polymerizing various functional monomers and a cross-linking agent in different components, and the affinity and the selectivity of the artificial antibody to the pathological marker of the Alzheimer's disease are regulated and controlled by changing the proportion of the functional monomers. By combining a molecular imprinting technology and taking the total protein or stable polypeptide fragment of the marker as a template, the specificity and selectivity of the nano artificial antibody to the pathological marker of the Alzheimer's disease are further improved. Testing the capability of the synthesized nano artificial antibody for effectively capturing the pathological marker of the Alzheimer's disease by utilizing a biomembrane interference technology, wherein the affinity constant K of the nano artificial antibody to the markerDThe value can reach 8.26 multiplied by 10-10M, comparable to antibodies, and excellent selectivity. The obtained nano artificial antibody can be combined with magnetic nano particles, an integral column or a microfluidic chip to realize high selective enrichment of low-abundance pathological markers of the Alzheimer's disease in a serum sample, and can realize high-sensitivity detection of the pathological markers of the Alzheimer's disease by methods such as high-sensitivity Raman spectroscopy, fluorescent quantitative detection, ELISA (enzyme-linked immunosorbent assay) kits, chemiluminescence kits and the like.
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 (6)
1. A preparation method of a non-biological nano artificial antibody of a specific targeting Alzheimer's disease marker is characterized in that N-isopropylacrylamide, N-tert-butylacrylamide, sodium dodecyl sulfate, a charged functional monomer, N, N ' -methylenebisacrylamide as a cross-linking agent and an Alzheimer's disease pathological marker as a template molecule are polymerized in a water phase under the action of an initiator and then eluted to obtain the non-biological nano artificial antibody;
the pathological markers of Alzheimer's disease comprise beta-amyloid, Tau protein, hyperphosphorylated protein holoprotein, or stable polypeptide fragments thereof;
the template molecule elution solution is any one of NaCl, sodium citrate, glycine or sodium dodecyl sulfate, or the elution of the template molecule is realized by changing the temperature and the pH value;
the charged functional monomer comprises any one or more of N- (3-aminopropyl) methacrylic acid, acrylic acid, methacrylic acid, 1-vinyl imidazole, N- (3-dimethylaminopropyl) methacrylamide, (3-acrylamidopropyl) trimethyl ammonium chloride or N- (2-aminoethyl) acrylamide;
the dosage of the N-isopropyl acrylamide is 5-60 wt%, the dosage of the N-tertiary butyl acrylamide is 5-50 wt%, the dosage of the charged functional monomer is 0.5-20 wt%, and the dosage of the N, N' -methylene bisacrylamide is 0.5-10 wt%.
2. The method for preparing the abiotic nano artificial antibody specifically targeting the Alzheimer's disease marker according to claim 1, wherein the polymerization method is inverse emulsion polymerization or precipitation polymerization.
3. The method for preparing the non-biological nano artificial antibody specifically targeting the Alzheimer's disease marker according to claim 1, wherein the initiator is ammonium persulfate or azobisisobutyronitrile.
4. The method for preparing the abiotic nano artificial antibody of the specific targeting Alzheimer's disease marker according to claim 2, wherein the polymerization temperature under nitrogen atmosphere is 20-70 ℃ and the polymerization time is 3-36 h.
5. The method for preparing the non-biological nano artificial antibody specifically targeting the Alzheimer's disease marker according to claim 1, wherein the particle size of the nano artificial antibody is 10-3000 nm.
6. Use of the non-biological nano artificial antibody specifically targeting the alzheimer's disease marker, characterized in that the non-biological nano artificial antibody specifically targeting the alzheimer's disease marker prepared according to the method of any one of claims 1 to 5 is used for realizing high selective enrichment of low abundance pathological markers of alzheimer's disease in serum samples in combination with magnetic nanoparticles, monolithic columns or microfluidic chips; the obtained nano artificial antibody realizes high-sensitivity detection of the pathological marker of the Alzheimer's disease by a high-sensitivity Raman spectrum, fluorescent quantitative detection, an ELISA kit, a chemiluminescence kit, a test strip, an electrochemical sensor, immunoturbidity, immunochromatography, ultrasonic detection, CT detection and nuclear magnetic detection method.
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