CN117753376A - Low-density lipoprotein cholesterol imprinting nanosphere and preparation method and application thereof - Google Patents
Low-density lipoprotein cholesterol imprinting nanosphere and preparation method and application thereof Download PDFInfo
- Publication number
- CN117753376A CN117753376A CN202311726060.7A CN202311726060A CN117753376A CN 117753376 A CN117753376 A CN 117753376A CN 202311726060 A CN202311726060 A CN 202311726060A CN 117753376 A CN117753376 A CN 117753376A
- Authority
- CN
- China
- Prior art keywords
- density lipoprotein
- low
- lipoprotein cholesterol
- nanospheres
- imprinted
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010028554 LDL Cholesterol Proteins 0.000 title claims abstract description 68
- 239000002077 nanosphere Substances 0.000 title claims abstract description 65
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 30
- 229920001690 polydopamine Polymers 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 19
- 238000003756 stirring Methods 0.000 claims abstract description 19
- 238000000926 separation method Methods 0.000 claims abstract description 18
- 229920000428 triblock copolymer Polymers 0.000 claims abstract description 17
- -1 polyethylene Polymers 0.000 claims abstract description 16
- CTENFNNZBMHDDG-UHFFFAOYSA-N Dopamine hydrochloride Chemical compound Cl.NCCC1=CC=C(O)C(O)=C1 CTENFNNZBMHDDG-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229960001149 dopamine hydrochloride Drugs 0.000 claims abstract description 15
- 238000005406 washing Methods 0.000 claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 12
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 12
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims abstract description 10
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 9
- 239000000839 emulsion Substances 0.000 claims abstract description 9
- 239000004698 Polyethylene Substances 0.000 claims abstract description 6
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 6
- 239000004743 Polypropylene Substances 0.000 claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims abstract description 6
- 229920000573 polyethylene Polymers 0.000 claims abstract description 6
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 6
- 229920001155 polypropylene Polymers 0.000 claims abstract description 6
- 238000004873 anchoring Methods 0.000 claims abstract description 5
- 239000012046 mixed solvent Substances 0.000 claims abstract description 5
- 239000007853 buffer solution Substances 0.000 claims abstract description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 30
- 239000000872 buffer Substances 0.000 claims description 7
- 238000010521 absorption reaction Methods 0.000 claims description 3
- AUHZEENZYGFFBQ-UHFFFAOYSA-N mesitylene Substances CC1=CC(C)=CC(C)=C1 AUHZEENZYGFFBQ-UHFFFAOYSA-N 0.000 claims description 3
- 125000001827 mesitylenyl group Chemical group [H]C1=C(C(*)=C(C([H])=C1C([H])([H])[H])C([H])([H])[H])C([H])([H])[H] 0.000 claims description 3
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 108010007622 LDL Lipoproteins Proteins 0.000 abstract description 44
- 102000007330 LDL Lipoproteins Human genes 0.000 abstract description 44
- 230000008569 process Effects 0.000 abstract description 4
- 238000000746 purification Methods 0.000 abstract description 3
- 238000001179 sorption measurement Methods 0.000 description 27
- 238000001000 micrograph Methods 0.000 description 10
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241000283707 Capra Species 0.000 description 5
- 230000006399 behavior Effects 0.000 description 5
- 230000005540 biological transmission Effects 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 229910052739 hydrogen Inorganic materials 0.000 description 5
- 239000001257 hydrogen Substances 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 235000012000 cholesterol Nutrition 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000002336 sorption--desorption measurement Methods 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- 208000024172 Cardiovascular disease Diseases 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000027455 binding Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000004580 weight loss Effects 0.000 description 3
- 230000017531 blood circulation Effects 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 1
- 206010070840 Gastrointestinal tract irritation Diseases 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 208000035150 Hypercholesterolemia Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 206010039020 Rhabdomyolysis Diseases 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 239000000370 acceptor Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000003763 carbonization Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 229960003638 dopamine Drugs 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000003446 memory effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000483 muscle toxicity Toxicity 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000001757 thermogravimetry curve Methods 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a low-density lipoprotein cholesterol imprinting nanosphere and a preparation method and application thereof, wherein a polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, an ethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer F127, dopamine hydrochloride and a pore-expanding agent are dispersed in a mixed solvent of ethanol and water to form an emulsion, ammonia water is added into the emulsion, and then the emulsion is stirred, and the porous polydopamine nanosphere is obtained through centrifugal separation and washing; dispersing in PBS buffer solution, adding low-density lipoprotein cholesterol, stirring at room temperature for surface anchoring, adding dopamine hydrochloride, stirring for reaction, washing after the reaction is finished to remove template protein, and obtaining the low-density lipoprotein cholesterol imprinted nanospheres. The low-density lipoprotein cholesterol imprinting nanospheres prepared by the invention can efficiently and specifically adsorb target LDL, and solve the problems of high requirements on equipment, complicated process and the like in the separation and purification of LDL.
Description
Technical Field
The invention belongs to the technical field of biological separation, and particularly relates to a low-density lipoprotein cholesterol imprinting nanosphere as well as a preparation method and application thereof.
Background
Low density lipoprotein cholesterol (Low-Density Lipoprotein Cholesterol, LDL-C), also known as Low density lipoprotein (Low Density Lipoprotein, LDL), refers to lipoproteins that are rich in cholesterol and esters thereof. When the human body ingests food containing cholesterol, cholesterol is decomposed by the liver into LDL-C, which is distributed throughout the body through blood circulation. LDL-C is a controllable factor in cardiovascular disease, and high levels of LDL-C can lead to the deposition and oxidation of cholesterol in blood vessels, forming atherosclerosis, thereby increasing the risk of cardiovascular disease. There is continued interest in how to reduce LDL-C levels.
The reduction of LDL-C levels, typically assessed by their concentration, has been considered a primary therapeutic goal for the prevention of primary and secondary cardiovascular disease for many years. Studies have shown that relying solely on diet and drugs to reduce LDL-C levels is not always effective. On the one hand many patients suffering from hypercholesterolemia due to gene mutation have not reached the reference low-density lipoprotein cholesterol concentration range, although the LDL-C lowering drug is fully used and the lifestyle is appropriately changed. On the other hand, long-term use of statins may lead to a number of serious adverse effects such as liver injury caused by elevated liver enzymes, muscle toxicity, gastrointestinal irritation and rhabdomyolysis. Thus, there is a need for lowering LDL-C levels by drug alternatives. The in vitro separation of LDL-C refers to a method for adsorbing and separating LDL-C in blood circulation by using an adsorbent, and the existing in vitro separation methods of LDL-C, such as a plasma replacement method, a double filtration method, an immunoadsorption method, a dextran sulfate adsorption method and a heparin-induced in vitro precipitation method, all need to separate plasma from whole blood through a plasma separation device before separating LDL-C from the plasma, so that the requirement on equipment is high and the process is complicated.
Disclosure of Invention
Aiming at the problems of higher equipment requirement and complicated separation process of the existing LDL-C separation and purification method, the invention provides the low-density lipoprotein cholesterol imprinting nanosphere, and the preparation method and application thereof, thereby realizing high-selectivity separation of LDL-C, simplifying the separation process, reducing the separation cost and improving the separation capacity.
The invention is realized by the following technical scheme:
the preparation method of the low-density lipoprotein cholesterol imprinting nanospheres comprises the following steps:
(1) Preparation of porous polydopamine nanospheres: dispersing a polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, an ethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer F127, dopamine hydrochloride and a pore-expanding agent in a mixed solvent of ethanol and water to form an emulsion, adding ammonia water into the emulsion, stirring, and carrying out centrifugal separation and washing to obtain porous polydopamine nanospheres;
(2) Preparation of low-density lipoprotein cholesterol imprinted nanospheres: dispersing the porous polydopamine nanospheres prepared in the step (1) in PBS buffer solution, adding low-density lipoprotein cholesterol, stirring at room temperature for surface anchoring, adding dopamine hydrochloride, stirring for reaction, and washing to remove template proteins after the reaction is finished to obtain the low-density lipoprotein cholesterol imprinted nanospheres.
Further, the ratio of the polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, the ethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer F127 and the dopamine hydrochloride in the step (1) is 1: 2-4: 4-7; the volume ratio of water to ethanol in the mixed solvent is 1: 0.5-2.
Further, in the step (1), the pore-expanding agent is mesitylene, and the addition amount of the pore-expanding agent and the volume mass of the ethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123 are 1mg: 15-20 mu L; the mass percentage concentration of the ammonia water is 20-30%, and the volume mass ratio of the added amount of the ammonia water to the ethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123 is 1mg:10 to 20 mu L.
Further, in the step (2), the mass ratio of the porous polydopamine nanospheres to the low-density lipoprotein cholesterol is 15-25: 1, the mass ratio of the porous polydopamine nanospheres to the dopamine hydrochloride is 1.5-3: 1.
further, adding ammonia water in the step (1), and stirring for 1.5-3 hours; in the step (2), stirring is carried out for 30-60 min at room temperature, and stirring reaction time is 14-20 h.
Further, the PBS buffer concentration in step (2) was 0.01 mol L -1 The pH was 8.5.
Further, washing the product after centrifugal separation in the step (1) for a plurality of times by adopting water and ethanol; the method of washing to remove the template protein in the step (2) is to wash with acetic acid a plurality of times to remove the template protein until the absorption peak cannot be detected by the ultraviolet-visible spectrophotometer at a wavelength of about 280 nm, and then wash with water and ethanol three times to remove the residual acetic acid.
Further, the acetic acid is an ethanol aqueous solution with the volume ratio of 15-30%.
In the invention, the low-density lipoprotein cholesterol imprinted nanospheres prepared by the preparation method are provided.
The invention discloses an application of a low-density lipoprotein cholesterol imprinted nanosphere in separating low-density lipoprotein cholesterol.
The beneficial effects of the invention are as follows:
the invention polymerizes triblock polymer P123, triblock copolymer F127, dopamine hydrochloride and pore-expanding agent under certain conditions to form porous polydopamine nanospheres as imprinting substrates, two similar block copolymers (P123 and F127) have hydrophilic chains with different lengths as soft templates, and mesoporous polydopamine nanoparticles are prepared by adopting a double soft template strategy; subsequently adding LDL, and attaching the template molecule LDL on the surface of the PPDA through pi-pi interaction, multiple hydrogen bonds and hydrophobic interaction based on protein anchoring technology to form PPDA-LDL complex; then adding dopamine hydrochloride, carrying out polymerization reaction under a certain condition to form a copolymer, eluting the copolymer, and forming a imprinting cavity complementary with the shape, the functional group and the space size of a template molecule on the surface of the imprinting polymer, wherein the PPDA-MIPs show excellent selectivity to LDL due to the shape memory effect of the imprinting cavity in recognition selectivity. The low-density lipoprotein cholesterol imprinting nanospheres prepared by the invention can efficiently and specifically adsorb target LDL, and solve the problems of high requirements on equipment, complicated process and the like in the separation and purification of LDL.
Drawings
FIG. 1 is a scanning electron microscope image of PPDA (a), PPDA-NIPs (b) and PPDA-MIPs (c); transmission electron microscope images of PPDA (d), PPDA-NIPs (e) and PPDA-MIPs (f);
FIG. 2 is an infrared spectrum (a) of PPDA, PPDA-MIPs and PPDA-NIPs and a thermogravimetric curve (b) of PPDA and PPDA-MIPs;
FIG. 3 shows the pH-dependent adsorption behavior (a) of LDL on the surface of PPDA, PPDA-NIPs and PPDA-MIPs and in the range of 0.0 to 3.0 mol L -1 A graph (b) of the relationship between LDL adsorption efficiency and ionic strength in the NaCl concentration range;
FIG. 4 shows adsorption isotherms (a) and 1/Q of LDL by PPDA-NIPs and PPDA-MIPs e For 1/C e Curve (b) of (a);
FIG. 5 shows the reusability of PPDA-MIPs in LDL separation (a) and SDS-PAGE analysis (b);
Detailed Description
The following description of the present invention is provided with reference to the accompanying drawings, but is not limited to the following description, and any modifications or equivalent substitutions of the present invention should be included in the scope of the present invention without departing from the spirit and scope of the present invention.
Example 1
(1) Preparation of porous polydopamine nanospheres (PPDA): 50 mg polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, 150 mg ethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer F127, 300 mg dopamine hydrochloride and 800 mu L of pore-enlarging agent mesitylene TMB are dispersed in a mixture of 10 mL ultrapure water and 10 mL ethanol, dispersed in an ultrasonic reactor for 5 min to form an emulsion, 750 mu L of ammonia water is added into the emulsion, stirring is carried out at room temperature for 2 h, and the porous polydopamine nanospheres (PPDA) are obtained by centrifugation and washing with ultrapure water and ethanol for a plurality of times;
(2) Preparation of Low Density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs): 100 mg of PPDA was dispersed in 50 mL of PBS buffer at pH 8.5 (PBS buffer concentration 0.01 mol L) -1 ) Then LDL of 5 mg was added. After stirring at room temperature for 50 min for surface anchoring, adding 50 mg dopamine hydrochloride into the solution, stirring for 16 h, washing with acetic acid (20% V/V) for multiple times to remove template proteins until an absorption peak cannot be detected at a wavelength of about 280 nm by an ultraviolet-visible spectrophotometer, and then washing with ultrapure water and ethanol for three times respectively to remove residual acetic acid, thereby obtaining the low-density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs).
Comparative example 1
(1) The same as in step (1) of example 1;
(2) Preparation of Low Density lipoprotein cholesterol non-imprinted nanospheres (PPDA-NIPs): 100 mg of PPDA was dispersed in 50 mL of PBS buffer at pH 8.5 (PBS buffer concentration 0.01 mol L) -1 ) Is a kind of medium. After stirring at room temperature for 50 min, 50 mg dopamine hydrochloride is added into the solution, stirring is continued for 16 h, then acetic acid (20% V/V) is used for washing for a plurality of times, and then ultrapure water and ethanol are used for washing for three times respectively, so that residual acetic acid is removed, and the low-density lipoprotein cholesterol non-imprinted nanospheres (PPDA-NIPs) are obtained.
The scanning electron microscope image and the transmission electron microscope image of the porous polydopamine nanospheres (PPDA), the low-density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) and the low-density lipoprotein cholesterol non-imprinted nanospheres (PPDA-NIPs) prepared in comparative example 1 are shown in fig. 1; wherein, (a) is a scanning electron microscope image of PPDA, (b) is a scanning electron microscope image of PPDA-MIPs, (c) is a scanning electron microscope image of PPDA-NIPs, (d) is a transmission electron microscope image of PPDA, (e) is a scanning electron microscope image of PPDA-MIPs, and (f) is a transmission electron microscope image of PPDA-NIPs. FIG. 1 (a) clearly shows that the prepared PPDA is a uniform nanosphere with a dense mesoporous structure, with a diameter of about 270 nm, and that the TEM image of 1 (d) further characterizes the rich mesoporous structure of PPDA, which provides sufficient specific surface area. Furthermore, it can be seen from the scanning electron microscope (FIGS. 1b and 1 c) and the transmission electron microscope (FIGS. 1e and 1 f) that the polymerization of dopamine results in PPDA-NIPs and PPDA-MIPs having a relatively rough surface structure and nano morphology.
The infrared spectroscopic images of the porous polydopamine nanospheres (PPDA), the low-density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) and the low-density lipoprotein cholesterol non-imprinted nanospheres (PPDA-NIPs) prepared in comparative example 1 are shown in fig. 2 (a); the spectrum of PPDA is about 3420. 3420 cm -1 There is a plate-like band, which is attributable to O-H stretching by intermolecular hydrogen bonding; at 1617, 1450 and 1114, 1114 cm -1 There is a set of distinguishable bands attributable to aromatic ring stretching vibrations of the polybenzazole structure 25; in addition, at 1496 and 1496 cm -1 The band of (2) corresponds to the N-H stretching vibration, at 1350 and 1292 cm -1 Typical peaks at the positions are respectively attributed to flexural vibration and tensile vibration of C-O-H on the benzene ring; notably, the FTIR spectra of PPDA-NIPs and PPDA-MIPs were similar to that of PPDA, confirming successful formation of polydopamine-based nanomaterials.
Thermogravimetric analysis curves of porous polydopamine nanospheres (PPDA), low density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) in example 1 are shown in fig. 2 (b); for PPDA and PPDA-MIPs, the first weight loss that occurs at 25-150 ℃ is a response to physically adsorbing water. For PPDA, a second weight loss of 45.4% can be noted due to the carbonization process of PPDA. The thermal behavior of PPDA-MIPs is similar to PPDA, but the second stage weight loss rate is as high as 48.8% higher than that of PPDA, which is related to the surface coating of PPDA with polydopamine.
Application examples
1. The porous polydopamine nanospheres (PPDA), low density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) and low density lipoprotein cholesterol non-imprinted nanospheres (PPDA-NIPs) in comparative example 1 were studied at different pH bars at pH 3-9Under-part pair LDL (15 μg mL) -1 300 μl) and the result is shown in fig. 3 (a), the adsorption efficiency of LDL on the surface of PPDA-MIPs is always higher than those of materials without blotting at pH 3-9. Meanwhile, the adsorption efficiency of LDL increases with increasing pH, and reaches a maximum at its isoelectric point (pH 5.5), while increasing the pH further decreases the adsorption efficiency of LDL. At pH 5.5, the optimal adsorption efficiencies of PPDA, PPDA-NIPs and PPDA-MIPs on LDL were 40.0%, 51.5% and 92.0%, respectively. The hydrogen bonding interactions between polydopamine nanospheres and low density lipoproteins can well explain this adsorption behavior. When the pH value is close to the pI of LDL, LDL is neutral, and electrostatic repulsive force is minimum. The chemical structure of polydopamine includes a number of hydrophilic groups including catechol, amine and imine, which can act as donors/acceptors for hydrogen bonding. Based on this, hydrogen bonding interactions result in maximum adsorption at pH values near pI of LDL.
2. The effect of low density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) in example 1 on LDL adsorption at different NaCl concentrations was studied, and the results are shown in FIG. 3 (b), which shows that the concentration of the low density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) is within a wide range (0 to 3 mol L -1 ) The change in ionic strength has no effect on the adsorption of LDL, indicating that electrostatic interactions do not contribute to the adsorption of LDL, demonstrating the utility of low-density lipoprotein cholesterol imprinted nanospheres in processing real biological samples that typically encounter relatively high ionic strengths.
3. 1-100 mug mL -1 The adsorption capacity of low-density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) in example 1 and low-density lipoprotein cholesterol non-imprinted nanospheres (PPDA-NIPs) in comparative example 1 to LDL was studied within the initial concentration range of (a), and the adsorption isotherm thereof was shown in fig. 4 (a), and the binding amount of LDL to PPDA-NIPs and PPDA-MIPs was rapidly increased with the increase of the initial LDL concentration and reached adsorption equilibrium. Under the same conditions, the maximum adsorption capacity of the PPDA-NIPs is 250.4 g mg -1 Whereas the maximum adsorption capacity of PPDA-MIPs is 550.3 g mg -1 . It follows that binding of LDL to PPDA-MIPs is mainly due to specific binding to recognition cavities. In contrast, the adsorption of LDL on PPDA-NIPs is predominantly non-specificActing as a medicine. To further analyze the adsorption behavior of PPDA-NIPs and PPDA-MIPs, a Langmuir isotherm model was applied to the equilibrium data. The equation is shown in formula (1):
formula (1)
C e （μg mL -1 ) Equilibrium concentration for LDL; q (Q) max （μg mg -1 ) And Q e （μg mg -1 ) The linear relationship between the maximum theoretical adsorption capacity and equilibrium adsorption capacity is shown in FIG. 4 (b), and the Langmuir model is suitable for the adsorption behaviors of PPDA-NIPs and PPDA-MIPs, and the correlation coefficients (R2) are 0.9900 and 0.9906, respectively. Thus, it is inferred that adsorption of LDL to the surface of a material is single-layered.
4. Elution and reuse of molecularly imprinted nanospheres
Reusability is an important index for evaluating the performance of molecularly imprinted materials. In the present invention, a desorption study was performed with MeOH: HAc (80:20) as eluent for 30 min, in the first adsorption/desorption cycle, 77.77% of adsorbed LDL was desorbed by PPDA-MIPs. Then, six adsorption/desorption cycles were repeated using the same blotting material (low density lipoprotein cholesterol blotting nanospheres (PPDA-MIPs) in example 1) under the optimal parameters, and as a result, as shown in fig. 5 (a), PPDA-MIPs were reduced in adsorption efficiency by only 11.51% after completion of 6 adsorption/desorption cycles, since part of the blotting sites were blocked by residual LDL and few binding sites were destroyed under acidic conditions. After 6 adsorption/desorption cycles, the adsorption efficiency can still reach 83.09%, which shows that the PPDA-MIPs have good recycling prospect.
5. Molecular imprinting nanosphere for separating and purifying LDL in goat serum sample
The low density lipoprotein cholesterol imprinted nanospheres (PPDA-MIPs) of example 1 were used for selective isolation of LDL in goat serum samples. With 4.0 mmol L -1 After goat serum was diluted 20-fold with BR buffer at pH 5.5, LDL was collected from PPDA-MIPs using MeOH in HAc (4:1) as eluent, ten samples were takenThe analysis was performed by sodium dialkylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the SDS-PAGE analysis was shown in FIG. 5 (b), from which several major bands were observed in the diluted goat serum samples. LDL having a molecular weight of about 2.7x10 3 -3.3x10 3 kDa, the position of the LDL standard solution is above the standard protein band and has a molecular weight of 200 kDa. Is identified at the same site as the LDL standard solution (lane 5). After adsorption of PPDA-MIPs (lane 3), the LDL bands clearly fade in color. In Lane 4, the PPDA-MIPs eluate showed a strong band above 200 kDa, and no other protein bands in the eluate, indicating good selectivity of PPDA-MIPs. The above results indicate that the prepared PPDA-MIPs can selectively recognize LDL in the presence of other proteins in goat serum samples.
Claims (10)
1. The preparation method of the low-density lipoprotein cholesterol imprinting nanospheres is characterized by comprising the following steps of:
(1) Preparation of porous polydopamine nanospheres: dispersing a polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, an ethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer F127, dopamine hydrochloride and a pore-expanding agent in a mixed solvent of ethanol and water to form an emulsion, adding ammonia water into the emulsion, stirring, and carrying out centrifugal separation and washing to obtain porous polydopamine nanospheres;
(2) Preparation of low-density lipoprotein cholesterol imprinted nanospheres: dispersing the porous polydopamine nanospheres prepared in the step (1) in PBS buffer solution, adding low-density lipoprotein cholesterol, stirring at room temperature for surface anchoring, adding dopamine hydrochloride, stirring for reaction, and washing to remove template proteins after the reaction is finished to obtain the low-density lipoprotein cholesterol imprinted nanospheres.
2. The method for preparing the low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, wherein the ratio of polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, ethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer F127 and dopamine hydrochloride in step (1) is 1: 2-4: 4-7; the volume ratio of water to ethanol in the mixed solvent is 1: 0.5-2.
3. The method for preparing the low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, wherein in the step (1), the pore-expanding agent is mesitylene, and the addition amount of the pore-expanding agent and the volume mass of the ethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123 are 1mg: 15-20 mu L; the mass percentage concentration of the ammonia water is 20-30%, and the volume mass ratio of the added amount of the ammonia water to the ethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123 is 1mg:10 to 20 mu L.
4. The method for preparing the low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, wherein the mass ratio of the porous polydopamine nanospheres to the low-density lipoprotein cholesterol in the step (2) is 15-25: 1, the mass ratio of the porous polydopamine nanospheres to the dopamine hydrochloride is 1.5-3: 1.
5. the method for preparing the low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, wherein the stirring time after adding ammonia water in the step (1) is 1.5-3 hours; in the step (2), stirring is carried out for 30-60 min at room temperature, and stirring reaction time is 14-20 h.
6. The method for preparing the low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, wherein the concentration of the PBS buffer in the step (2) is 0.01 mol L -1 The pH was 8.5.
7. The method for preparing the low-density lipoprotein cholesterol imprinted nanospheres according to claim 1, wherein the product after centrifugal separation in the step (1) is washed with water and ethanol for several times; the method of washing to remove the template protein in the step (2) is to wash with acetic acid a plurality of times to remove the template protein until the absorption peak cannot be detected by the ultraviolet-visible spectrophotometer at a wavelength of about 280 nm, and then wash with water and ethanol three times to remove the residual acetic acid.
8. The method for preparing the low-density lipoprotein cholesterol imprinted nanospheres according to claim 7, wherein the acetic acid is an ethanol aqueous solution with a volume ratio of 15-30%.
9. A low-density lipoprotein cholesterol imprinted nanosphere prepared by the preparation method of any one of claims 1 to 8.
10. Use of the low-density lipoprotein cholesterol imprinted nanospheres of claim 9 for separating low-density lipoprotein cholesterol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311726060.7A CN117753376A (en) | 2023-12-15 | 2023-12-15 | Low-density lipoprotein cholesterol imprinting nanosphere and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311726060.7A CN117753376A (en) | 2023-12-15 | 2023-12-15 | Low-density lipoprotein cholesterol imprinting nanosphere and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117753376A true CN117753376A (en) | 2024-03-26 |
Family
ID=90323050
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311726060.7A Pending CN117753376A (en) | 2023-12-15 | 2023-12-15 | Low-density lipoprotein cholesterol imprinting nanosphere and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117753376A (en) |
-
2023
- 2023-12-15 CN CN202311726060.7A patent/CN117753376A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tsukagoshi et al. | Metal Ion-Selective Adsorbent Prepared by Surface-Imprinting Polymerization. | |
| KR100861452B1 (en) | Method for preparing surface-imprinted polyacrylate microsphere in the form of core-shell for the selective separation of heavy metal ion | |
| Liu et al. | Tailored Janus silica nanosheets integrating bispecific artificial receptors for simultaneous adsorption of 2, 6-dichlorophenol and Pb (ii) | |
| Zhang et al. | Fabrication and evaluation of molecularly imprinted magnetic nanoparticles for selective recognition and magnetic separation of lysozyme in human urine | |
| Tarasi et al. | Laccase immobilization onto magnetic β-cyclodextrin-modified chitosan: improved enzyme stability and efficient performance for phenolic compounds elimination | |
| CN108912269B (en) | Polyimidazole ionic liquid-based copolymer and preparation method and application thereof | |
| Yuan et al. | A novel ionic liquid polymer material with high binding capacity for proteins | |
| CN112774642B (en) | Preparation method of nano magnetic adsorbent with selective adsorption function on Pb (II) | |
| EP2075276A1 (en) | Method for producing a sorbent based on a methyl- silicic acid hydrogel | |
| Parvinizadeh et al. | Fabrication of a magnetic metal–organic framework molecularly imprinted polymer for extraction of anti-malaria agent hydroxychloroquine | |
| Wang et al. | Construction of copper (II) affinity-DTPA functionalized magnetic composite for efficient adsorption and specific separation of bovine hemoglobin from bovine serum | |
| CN111974366A (en) | Preparation and application of amphoteric carboxymethyl chitosan-based microspheres based on magnetic separation technology | |
| Xu et al. | Preparation and characterization of surface molecularly imprinted films coated on multiwall carbon nanotubes for recognition and separation of lysozyme with high binding capacity and selectivity | |
| Luan et al. | Construction of magnetic covalent organic frameworks functionalized by benzoboroxole for efficient enrichment of glycoproteins in the physiological environment | |
| Arabzadeh et al. | Synthesis and characterization of molecularly imprinted polymers for selective solid-phase extraction of pseudoephedrine | |
| Ding et al. | Preparation and characterization of biocompatible molecularly imprinted poly (ionic liquid) films on the surface of multi-walled carbon nanotubes | |
| Xu et al. | Preparation of biocompatible molecularly imprinted film on biowaste-derived magnetic pomegranate rind carbon for protein recognition in biological sample | |
| Wu et al. | Poly (aspartic acid) surface modification of macroporous poly (glycidyl methacrylate) microspheres | |
| CN109650602B (en) | Method for removing antimony ions in water body by using magnetic adsorption material | |
| US10435486B2 (en) | Polymers prepared using smart templates | |
| Liu et al. | Protein imprinting over magnetic nanospheres via a surface grafted polymer for specific capture of hemoglobin | |
| CN117753376A (en) | Low-density lipoprotein cholesterol imprinting nanosphere and preparation method and application thereof | |
| CN109225341B (en) | Preparation method and application of visible light response magnetic metal organic framework material | |
| Wen et al. | Fabrication of hyperbranched polyamidoamine immobilized mesocellular siliceous foam for rapid decontamination of lead (II) from aqueous solution | |
| Xu et al. | Super-adsorbent microspheres based on a triallyl isocyanurate–maleic anhydride copolymer for the removal of organic pollutants from water |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |