CN117849331A - Detection reagent for N-phosphorylation modified biomolecules and preparation method and application thereof - Google Patents
Detection reagent for N-phosphorylation modified biomolecules and preparation method and application thereof Download PDFInfo
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- CN117849331A CN117849331A CN202410018450.7A CN202410018450A CN117849331A CN 117849331 A CN117849331 A CN 117849331A CN 202410018450 A CN202410018450 A CN 202410018450A CN 117849331 A CN117849331 A CN 117849331A
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Abstract
The invention belongs to the field of functionalized high polymer materials, and particularly relates to a detection reagent for N-phosphorylation modified biomolecules, and a preparation method and application thereof. The detection reagent provided by the invention comprises a Pincer type bimetal functionalized dendritic polymer; the functionalized dendritic polymer is prepared from raw materials comprising an A component and a B component; the A component is a dendritic polymer; the component B is a piver type ligand-bimetallic coordination compound; the Picer type ligand-bimetallic coordination compound is a Picer type coordination-double zinc coordination compound or a Picer type coordination-double copper coordination compound. The invention can realize quantitative analysis of the total N-phosphorylation modification level of the N-phosphorylation modified biomolecules, and can be combined with a conventional reagent capable of realizing the specific detection of the biomolecules to be detected to realize high-sensitivity and high-specificity detection of the N-phosphorylation modification level.
Description
Technical Field
The invention belongs to the field of functionalized high polymer materials, and particularly relates to a detection reagent for N-phosphorylation modified biomolecules, and a preparation method and application thereof.
Background
Biomolecules are widely modified by N-phosphorylation, such as small N-phosphorylated biomolecules, N-phosphorylated proteins, N-phosphorylated peptides, etc., which play a role in important life processes such as cell signaling, metabolic regulation, immune function regulation, etc., and many biomolecules containing N-phosphorylated modifications are biomarkers or drug therapeutic targets for the occurrence and development of diseases and even cancers. Therefore, development of detection reagents or methods capable of directly detecting biomolecules containing N-phosphorylation modification is of great importance for early warning, targeted therapy and the like of cancers.
The detection difficulty of N-phosphorylation modified biomolecules is that conventional detection reagents for phosphorylation modification of biomolecules need to be capable of effectively recognizing the phosphorylation modification of the biomolecules under strong acid conditions, and the phosphorylation modification of N-phosphorylation modified biomolecules is unstable under strong acid conditions and is easy to dephosphorylate. Therefore, it has been a technical difficulty how to achieve specific detection of N-phosphorylation modified biomolecules under weak or near neutral conditions.
Currently, few specific detection means are available for N-phosphorylation modification, including mainly immunodetection, mass spectrometry and isotope methods. The immunodetection method needs to prepare high-specificity and high-affinity antibodies, and the N-phosphorylated proteins and N-phosphorylated amino acids are easy to dephosphorylate when used as antigens for immunizing experimental animals, so that effective antibodies are difficult to generate. Even though various N-phosphorylated amino acid analogs have been developed as antigens by researchers, most of the N-phosphorylated antibodies prepared can only recognize a single site, and different antibodies need to be developed for N-phosphorylation detection at different sites, so that a new method for detecting universal N-phosphorylation modification is urgently needed.
In recent years, methods for enriching N-phosphorylated proteins using affinity materials have been developed. For example, chinese patent CN111203185A, a dimedo picoline amine dimedo functional material is obtained by introducing dimedo picoline amine dimedo molecule on the surface of a mesoporous core-shell silicon sphere carrier, and the specificity enrichment, purification and separation of histidine phosphorylated peptide segments are successfully realized under weak acid conditions. The specific recognition principle of the dimetyl pyridinamine dimyristyl on the biomolecules containing N-phosphorylation modification is that the dimetyl pyridinamine dimyristyl functional molecules are formed by utilizing the synergistic effect of dimetyl pyridinamine groups and zinc ions; zinc ions in the dimedoxypyridinamine dimedoxyzinc functional molecule can be coordinated with phosphate groups of the N-phosphorylation modified biomolecules to realize the specific recognition of the N-phosphorylation modified biomolecules.
However, the dimedo picoline amine dimedo zinc functionalized mesoporous core-shell silica spheres are not bonded with detectable functional groups or antibody groups, or contain functional groups or antibody groups which can be combined with the detected functional groups, and the mesoporous core-shell silica spheres are solid-phase materials and can not be dissolved in water, so that the dimedo picoline amine dimedo zinc functionalized mesoporous core-shell silica spheres are difficult to be directly applied to detection technologies such as Western blot, SDS-PAGE and ELISA in biological sample detection as detection reagents.
Disclosure of Invention
The invention aims to provide a detection reagent for N-phosphorylation modified biomolecules, a preparation method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a detection reagent of N-phosphorylation modified biomolecules, which comprises a Pincer type bimetal functionalized dendritic polymer; the functionalized dendritic polymer is prepared from raw materials comprising an A component and a B component;
the A component is a dendritic polymer;
the component B is a piver type ligand-bimetallic coordination compound; the Picer type ligand-bimetallic coordination compound is a Picer type coordination-double zinc coordination compound or a Picer type coordination-double copper coordination compound.
Preferably, the functionalized dendrimer is further bonded with a C component, which is a functional group that can achieve direct detection or an antibody group that can achieve direct detection.
Preferably, the functionalized dendrimer is further bonded with a D component, which is a functional group that can be bound to a C component or an antibody group that can be bound to a C component.
Preferably, the dendritic polymer is a polyamide-amine type dendritic polymer or a co-embedded dendritic polymer of a polyamide-amine type dendritic polymer.
Preferably, the polyamide-amine type dendritic polymer is one or more of the second generation to the tenth generation.
Preferably, the component B is a Picer type coordination-bimetallic coordination compound containing heteroatom coordination, and is obtained by using a Picer type ligand containing heteroatom coordination and a metal ion;
the heteroatom comprises one or more of oxygen, nitrogen and sulfur;
the Picer type ligand containing heteroatom coordination comprises one or more of 2,2' -dimethylpyridine amine, 2' -dimethylpyrazole amine, 2' -dimethylpyrimidine amine, 2' -dimethylquinoline amine, 2' -dimethylthiazole amine, 2' -dimethylfuran amine and 2,2' -dimethylisoquinoline amine.
Preferably, the functional group in the component C capable of realizing direct detection comprises a fluorescent group or a luminescent group;
the functional group which can be combined with the C component in the D component comprises biotin, hapten or crosslinking group.
The invention also provides a preparation method of the detection reagent according to the scheme, which comprises the following steps:
(1) Mixing the dendritic polymer solution with a Picer type ligand solution and a metal ion solution for a coupling reaction to obtain a solution containing a Picer type bimetal functionalized dendritic polymer; the metal ion solution is zinc ion solution or copper ion solution;
(2) Purifying the solution containing the Pincer type bimetal functionalized dendritic polymer to obtain the detection reagent of the N-phosphorylation modified biological molecule.
Preferably, the preparation method of the Picancer type ligand solution comprises the following steps: mixing an amine compound, paraformaldehyde and a first solvent for a Mannich reaction; carrying out first amidation reaction on the obtained reaction product and N-t-butoxycarbonyl-L-tyrosine; purifying the obtained product, and then mixing the purified product with dichloromethane and trifluoroacetic acid to carry out a protective group removal reaction; carrying out a second amidation reaction on the obtained reaction product and an anhydride-containing dichloromethane solution to obtain a carboxyl-terminated four-arm amine compound; and mixing the carboxyl-terminated four-arm amine compound, dimethyl sulfoxide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide for performing a third amidation reaction to obtain a Picer type ligand solution.
The invention also provides application of the detection reagent according to the scheme or the detection reagent obtained by the preparation method according to the scheme in preparation of a kit for detecting N-phosphorylation modified biomolecules.
The invention provides a detection reagent for N-phosphorylation modified biomolecules. The invention uses dendritic polymer as matrix to support the functional group containing the Picer type ligand-bimetallic coordination compound. On one hand, the dendritic polymer has excellent water solubility, and the detection reagent of the N-phosphorylation modified biomolecules with the dendritic polymer as a matrix can realize the specific detection of the N-phosphorylation modified biomolecules in a water phase, thereby being beneficial to being directly applied to detection technologies such as Western blot, SDS-PAGE, ELISA and the like which are most widely applied in biological sample detection. On the other hand, the dendritic polymer has a unique hyperbranched structure and nanoscale, and the surface of the dendritic polymer contains dense surface active functional groups, so that simultaneous immobilization of a plurality of Picer type ligand-bimetallic coordination compound functional groups can be realized, the affinity of the detection reagent of the N-phosphorylation modified biomolecules to the N-phosphorylation modified biomolecules is improved, and the detection specificity of the N-phosphorylation modified biomolecules is improved. In addition, the functional group containing the Picer type ligand-bimetallic coordination compound can realize the specific recognition of the N-phosphorylation modified biological molecule under the weak acid or near neutral condition, and the N-phosphorylation modified biological molecule has high stability under the condition, so that the dephosphorylation of the N-phosphorylation modified biological molecule can be effectively avoided, and the detection sensitivity of the N-phosphorylation modified biological molecule is further effectively improved.
The invention can be directly used for detecting the biological molecules containing N-phosphorylation modification by using the detection reagent. (1) The invention can realize the quantitative analysis of the total N-phosphorylation modification level of all N-phosphorylation modified biomolecules in the sample to be detected. The invention uses dendritic polymer as matrix to support the functional group containing the Picer type ligand-bimetallic coordination compound. The dendrimer has excellent water solubility, and the detection reagent of the N-phosphorylation modified biomolecules with the dendrimer as a matrix can realize homogeneous incubation and efficient combination with samples containing the N-phosphorylation modified biomolecules in an aqueous phase. The functional group containing the Picer type ligand-bimetallic coordination compound immobilized on the detection reagent of the N-phosphorylation modified biomolecules can realize the selective identification of all N-phosphorylation modified biomolecules in the sample under weak acid or near neutral condition.
(2) The invention can be combined with a conventional reagent capable of realizing the specific detection of the biological molecule to realize the high-sensitivity and high-specificity detection of the target N-phosphorylation modified biological molecule. The dendritic polymer used in the invention has excellent water solubility, can be coated on a membrane (such as a polyvinylidene fluoride membrane, a nitrocellulose membrane and the like) or in an orifice plate (such as a common polypropylene micro-orifice plate) in an incubation mode, and the functional group containing the Picer type ligand-bimetallic coordination compound immobilized on the detection reagent of the N-phosphorylation modified biomolecules on the membrane or in the orifice plate can realize the specific capture of the N-phosphorylation modified biomolecules in an aqueous phase under weak acid or near neutral condition, and can realize the specific, high-sensitivity qualitative and quantitative detection of the N-phosphorylation modified level of the target N-phosphorylation modified biomolecules by using the conventional specific detection reagent or antibody of the target biomolecules after the non-N-phosphorylation modified biomolecules are removed by flushing.
The detection reagent provided by the invention has the following advantages: (1) high sensitivity: the detection limit of the detection reagent for the N-phosphorylation modified biomolecules provided by the invention on the N-phosphorylation modified biomolecules depends on the content of the N-phosphorylation modification of the biomolecules and the detection groups used in the detection reagent, and the detection reagent for the N-phosphorylation modified biomolecules provided by the invention can be used for detecting more than 1ng of N-phosphorylation modified biomolecules; (2) strong anti-interference capability: the detection reagent can selectively detect the biomolecules containing the N-phosphorylation modification under the interference of 100 times of biomolecules without the N-phosphorylation modification; (3) good stability: the detection reagent can be repeatedly used for a plurality of times, and can keep good performance within a storage period of up to several months if the detection reagent is stored properly; (4) the detection reagent has low cost and convenient use; (5) strong universality: the method is applicable to detection of N-phosphorylated biological samples containing different sites, and meets the universality requirement.
Further, the functionalized dendritic polymer is also bonded with a C component, wherein the C component is a functional group capable of realizing direct detection or an antibody group capable of realizing direct detection; the functionalized dendrimer is further bonded with a D component, which is a functional group that can be bound to a C component or an antibody group that can be bound to a C component. The invention realizes high-sensitivity quantitative analysis of the total N-phosphorylation modification level of the N-phosphorylation modified biomolecules in the sample on the basis of selectively identifying all N-phosphorylation modified biomolecules in the sample by the functional groups containing the Picer type ligand-bimetallic coordination compound through the component C and the component D.
The invention also provides a preparation method of the detection reagent of the N-phosphorylation modified biological molecule. The preparation method provided by the invention has the advantages of simple steps, strong anti-interference capability, good stability, low cost, mild condition and convenient operation.
The invention also provides an application of the detection reagent of the N-phosphorylation modified biomolecule in the scheme or the detection reagent of the N-phosphorylation modified biomolecule obtained by the preparation method in the scheme in preparation of a kit for detecting the N-phosphorylation modified biomolecule. The detection reagent provided by the invention is applied to quantitative analysis of the total N-phosphorylation modification level of all N-phosphorylation modified biomolecules in a sample to be detected, is simple and easy to operate, is very suitable for preparing a kit for detecting the N-phosphorylation modified biomolecules, and can also be used for popularization and popularization in the fields of analytical chemistry, inspection chemistry, biochemistry, food detection and the like.
The detection reagent of the N-phosphorylation modified biomolecules can be used for detecting the free N-phosphorylation modified biomolecules in solution (for example, applied to enzyme-linked immunosorbent assay (ELISA)), detecting the N-phosphorylation modified biomolecules bound on a carrier (for example, applied to immunoblotting, immunohistochemistry and other experiments), and detecting the N-phosphorylation modified biomolecules in gel (for example, polyacrylamide gel electrophoresis experiments).
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is an infrared signature of a detection reagent for a bis-picolinamine bis-zinc functionalized N-phosphorylation modified biomolecule of example 1 of the present invention; wherein (A) is a fourth generation polyamide-amine dendrimer, (B) is a bis-lutidine amine modified polyamide-amine dendrimer, (C) is a bis-lutidine amine bis-zinc functionalized polyamide-amine dendrimer;
FIG. 2 is a representation of the X-ray photoelectron spectroscopy (XPS) of zinc ions of the bis-picolinamine bis-zinc functionalized detection reagent for detecting N-phosphorylated modified biomolecules of example 1 of the present invention;
FIG. 3 shows the application of the detection reagent for detecting N-phosphorylation modified biomolecules functionalized with dimpyrilamine biszinc of example 1 to the spot-imprinting (Blotting) detection of N-phosphorylated proteins on Nitrocellulose (NC) membranes; wherein (A) is bovine serum albumin BSA and N-phosphorylated bovine serum albumin N-BSA, (B) is Casein beta-Casein and N-phosphorylated Casein N-beta-Casein;
FIG. 4 shows the application of the biotin-modified bis-dimethylpyrazolamide bis-zinc functionalized detection reagent for detecting N-phosphorylated biomolecules in the detection of spot-imprinting (Blotting) of N-phosphorylated proteins on polyvinylidene fluoride (PVDF) membranes in accordance with example 8 of the present invention; wherein, (A) is an image of non-N-phosphorylated protein (bovine serum albumin-BSA) and N-phosphorylated protein (N-phosphorylated bovine serum albumin-BSA) with different concentrations after chromogenic detection by the detection reagent of the present invention, and (B) is a linear response curve between the sample amount and OD value of the N-phosphorylated protein (N-phosphorylated bovine serum albumin-N-BSA) obtained by the detection reagent of the present invention;
FIG. 5 shows the application of the detection reagent for detecting N-phosphorylation modified biomolecules functionalized with dimpyrilamine biszinc in example 1 to dynamic real-time immunoblotting (WesternBlotting) detection of N-phosphorylation levels of N-phosphorylated proteins on polyvinylidene fluoride (PVDF) membranes; wherein, (A) is an image after the N-phospho horseradish peroxidase N-HRP is detected by the detection reagent of the invention, and (B) is a linear response curve between an OD value obtained by the N-phospho level of the N-phospho horseradish peroxidase N-HRP (N-HRP) obtained by the detection reagent of the invention and the N-phospho reaction time, and (C) is an image after the N-phospho myoglobin N-myoglobin is detected by the detection reagent of the invention; (D) A linear response curve between the OD value obtained for the N-phosphorylation level of N-phosphorylated myoglobin (N-myolobin) obtained using the detection reagent of the present invention and the N-phosphorylation reaction time;
FIG. 6 shows the reusability and stability of the biotin-modified bis-dimethylpyrazolamide bis-copper functionalized detection reagents for detecting N-phosphorylated biomolecules according to example 2 of the present invention when applied to immunoblotting (WesternBlotting) detection of N-phosphorylated proteins on polyvinylidene fluoride (PVDF) membranes; wherein, (A) is reusability when the detection reagent of the present invention is repeatedly applied to detection of different sample amounts of N-phosphorylated proteins (N-phosphorylated bovine serum albumin N-BSA and bovine serum albumin BSA) in an immunoblotting method three times, and (B) is stability when the detection reagent stored for different time periods is applied to detection of different sample amounts of N-phosphorylated proteins (N-phosphorylated bovine serum albumin N-BSA and bovine serum albumin BSA) in an immunoblotting method;
FIG. 7 shows the application of biotin-modified bis-dimethylpyrazolamide bis-zinc functionalized detection reagent for detecting N-phosphorylated biomolecules in ELISA detection of N-phosphorylated proteins in accordance with example 12 of the present invention; wherein (B) is a linear response curve between the sample amount and OD value of the N-phosphorylated protein (N-phosphorylated bovine serum albumin N-BSA) obtained using the detection reagent of the present invention.
Detailed Description
The invention provides a detection reagent of N-phosphorylation modified biomolecules, which comprises a Pincer type bimetal functionalized dendritic polymer; the functionalized dendritic polymer is prepared from raw materials comprising an A component and a B component;
the A component is a dendritic polymer;
the component B is a piver type ligand-bimetallic coordination compound; the Picer type ligand-bimetallic coordination compound is a Picer type coordination-double zinc coordination compound or a Picer type coordination-double copper coordination compound.
In the present invention, the a-component is a dendrimer; the dendritic polymer is preferably a polyamide-amine dendritic polymer or a co-embedded dendritic polymer of a polyamide-amine dendritic polymer; the polyamide-amine type dendritic polymer is preferably one or more of the second to tenth generations.
In the present invention, the B component is preferably a piter type ligand-bimetallic complex compound containing heteroatom coordination, derived from a piter type ligand containing heteroatom coordination and a metal ion; the heteroatom preferably includes one or more of oxygen, nitrogen and sulfur; the heteroatom-containing coordinated Picor type ligand preferably comprises one or more of 2,2' -dimethylpyridine amine, 2' -dimethylpyrazole amine, 2' -dimethylpyrimidine amine, 2' -dimethylquinoline amine, 2' -dimethylthiazole amine, 2' -dimethylfuran amine and 2,2' -dimethylisoquinoline amine.
In the present invention, the functionalized dendrimer is preferably further bonded with a C component, which is a functional group that can achieve direct detection or an antibody group that can achieve direct detection; the functional group capable of realizing direct detection in the component C comprises a fluorescent group or a luminescent group; the fluorescent group preferably comprises one or more of acridine orange, acridine yellow, 4', 6-diamidino-2-phenylindole, fluorescein, alexa Fluor series dye, cyanine series dye, rhodamine, texas red, phthalaldehyde, coumarin type dye, nile red, oregon green dye, propidium iodide, cascade blue, luciferase and green fluorescent protein; the luminescent group preferably comprises one or more of luminol, isoluminol, acridinium ester, and az amide, adamantane and derivatives thereof.
In the present invention, the functionalized dendrimer is preferably further bonded with a D component; the component D is a functional group capable of being combined with the component C or an antibody group capable of being combined with the component C; the functional group in the D component which can be combined with the C component preferably comprises biotin, hapten or crosslinking group; the crosslinking group preferably includes one or more of an aldehyde group, a carboxyl group and an amino group.
In the present invention, the biomolecules in the N-phosphorylation modified biomolecules preferably include polypeptides, proteins or biological small molecules; the biomolecule is preferably a basic amino acid containing biomolecule; the basic amino acid preferably comprises one or more of histidine, arginine and lysine; the N-phosphorylation modification preferably occurs on a side chain amino group of the basic amino acid.
The invention also provides a preparation method of the detection reagent according to the scheme, which comprises the following steps:
(1) Mixing the dendritic polymer solution with a Picer type ligand solution and a metal ion solution (marked as first mixing) for coupling reaction to obtain a solution containing a Picer type bimetal functionalized dendritic polymer; the metal ion solution is zinc ion solution or copper ion solution;
(2) Purifying the solution containing the Pincer type bimetal functionalized dendritic polymer to obtain the detection reagent of the N-phosphorylation modified biological molecule.
The invention mixes dendritic polymer solution with the Picer type ligand solution and metal ion solution (marked as first mixing) to carry out coupling reaction, thus obtaining the solution containing the Picer type bimetal functionalized dendritic polymer.
In the present invention, the preparation method of the piner type ligand solution is preferably as follows: mixing an amine compound, paraformaldehyde and a first solvent for a Mannich reaction; carrying out first amidation reaction on the obtained reaction product and N-t-butoxycarbonyl-L-tyrosine; purifying the obtained product, and then mixing the purified product with dichloromethane and trifluoroacetic acid to carry out a protective group removal reaction; carrying out a second amidation reaction on the obtained reaction product and an anhydride-containing dichloromethane solution to obtain a carboxyl-terminated four-arm amine compound; and mixing the carboxyl-terminated four-arm amine compound, dimethyl sulfoxide (DMSO), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) for performing a third amidation reaction to obtain a Picer type ligand solution.
In the present invention, the amine compound preferably includes one or more of 2,2' -dimethylpyridine amine, 2' -dimethylpyrazole amine, 2' -dimethylpyrimidine amine, 2' -dimethylquinoline amine, 2' -dimethylthiazole amine, 2' -dimethylfuran amine and 2,2' -dimethylisoquinoline amine; the first solvent is preferably an isopropyl alcohol/water mixed solution; the content of isopropanol in the isopropanol/water mixed solution is preferably 35%; the pH value of the isopropanol/water mixed solution is preferably 8.0; the mass ratio of the amine compound to the paraformaldehyde is preferably 1:1; the amount-to-volume ratio of the amine compound to the substance of the first solvent is preferably 5 mmol/1 mL; the temperature of the mannich reaction is preferably 80 ℃ and the incubation time is preferably 0.5 hours.
In the invention, the mass ratio of the amine compound to the N-t-butoxycarbonyl-L-tyrosine is preferably 1:1; the temperature of the first amidation reaction is preferably 110℃and the holding time is preferably 13 hours.
In the present invention, the purification is preferably silica gel column chromatography; the mass ratio of the amine compound to the dichloromethane is preferably 5:5 to 10, more preferably 5:6 to 9, and even more preferably 5:7 to 8; the amount-to-volume ratio of the amine compound to the trifluoroacetic acid is preferably 5 mmol/10 mL; the temperature of the protecting group removal reaction is preferably 30-40 ℃, more preferably 35 ℃, and the heat preservation time is preferably 6 hours.
In the present invention, the acid anhydride preferably includes one or both of succinic anhydride and glutaric anhydride; the concentration of the methylene chloride solution containing the anhydride is preferably 0.05mmol/mL; the mass ratio of the amine compound to the anhydride is preferably 5:5.1; the temperature of the second amidation reaction is preferably 50℃and the holding time is preferably 18 hours.
In the present invention, the mass to volume ratio of the carboxyl-terminated quaternary ammonium compound to dimethyl sulfoxide is preferably 5mg to 150. Mu.L; the mass ratio of the carboxyl-terminated four-arm amine compound to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is preferably 5:10; the ratio of the carboxyl-terminated four-arm amine compound to the N-hydroxysuccinimide is preferably 5:1; the temperature of the third amidation reaction is preferably room temperature, and the holding time is preferably 12 hours; the third amidation reaction is preferably performed under stirring.
In the present invention, the preparation method of the dendrimer solution is preferably: dissolving the dendritic polymer in absolute methanol to obtain a dendritic polymer solution (marked as a solution B); the mass to volume ratio of the dendrimer to the anhydrous methanol is preferably 2 mg/500. Mu.L.
In the present invention, the preparation method of the metal ion solution preferably comprises: zn (NO) 3 ) 2 ·6H 2 O or Cu (NO) 3 ) 2 ·6H 2 Mixing O with water to obtain a metal ion solution (marked as solution D); the water is preferably purified water; the Zn (NO) 3 ) 2 ·6H 2 O or Cu (NO) 3 ) 2 ·6H 2 The mass to volume ratio of O to water is preferably 20mg to 30. Mu.L.
In the present invention, the quantitative concentration ratio of the substance of the dendrimer solution to the substance of the piver type ligand solution is preferably 1.1:0.9; the volume ratio of the dendrimer solution to the metal ion solution is preferably 10:9.0.
In the present invention, the first mixture is preferably: the solution B and the solution a are mixed to obtain a solution C, and then the obtained solution C and the solution D are mixed.
In the present invention, the temperature of the first mixture is preferably room temperature, and the holding time is preferably 12 to 36 hours, more preferably 18 to 24 hours; the first mixing is preferably mechanical stirring.
In the present invention, the conditions of the first mixing are preferably: if the mixture to be mixed is solid or liquid and is not mutually soluble, the solvent is used for dissolving the mixture to be mixed, and if the mixture to be mixed is liquid and is mutually soluble, the mixture to be mixed is mixed.
In the present invention, the coupling reaction is preferably carried out in a constant temperature shaking table, the reaction temperature is preferably room temperature to 50 ℃, and the incubation time is preferably 24 hours.
After obtaining the solution containing the Piner type bimetal functionalized dendritic polymer, the invention purifies the solution containing the Piner type bimetal functionalized dendritic polymer (marked as first purification) to obtain the detection reagent of the N-phosphorylation modified biological molecule.
In the present invention, the first purifying apparatus is preferably an ultrafiltration membrane or a dialysis bag; the molecular weight cut-off of the ultrafiltration membrane or dialysis bag is preferably 3000Da.
In the present invention, the first purification preferably further comprises: mixing the obtained purified product with a C component or a D component (marked as a second mixture), performing Suzuki coupling reaction, and purifying (marked as a second purification) to obtain the detection reagent of the N-phosphorylation modified biological molecule.
In the present invention, the second mixing is preferably mechanical stirring; the temperature of the second mixing is preferably room temperature, and the mixing time is preferably 2 to 36 hours, more preferably 10 to 20 hours.
In the present invention, the second mixture is preferably: mixing the purified product with a phosphate solution (denoted as first phosphate solution) to obtain solution E; mixing the component C or the component D with a phosphate solution (named as second phosphate solution) to obtain a solution F; the solution E and the solution F were mixed.
In the present invention, the volume ratio of the purified product to the first phosphate solution is preferably 10:9.0 to 9.5, more preferably 10:9.0; the mass to volume ratio of the C or D component and the second phosphate solution is preferably (0.8-1) mg: 300. Mu.L, more preferably 0.9mg: 300. Mu.L.
In the invention, the temperature of the Suzuki coupling reaction is preferably room temperature to 60 ℃, and the heat preservation time is preferably 2 hours; the Suzuki coupling reaction is carried out under the condition of mechanical stirring.
In the present invention, the second purifying apparatus is preferably an ultrafiltration membrane or a dialysis bag; the molecular weight cut-off of the ultrafiltration membrane or dialysis bag is preferably 3000Da.
The invention also provides application of the detection reagent according to the scheme or the detection reagent obtained by the preparation method according to the scheme in preparation of a kit for detecting N-phosphorylation modified biomolecules.
The detection reagent is combined with a conventional reagent capable of realizing the specific detection of the biomolecules to be detected, so that the high-sensitivity and high-specificity detection of the N-phosphorylation modified biomolecules to be detected in a biological system is realized; the detection reagent can be applied to quantitative analysis of the total N-phosphorylation modification level of all N-phosphorylation modified biomolecules in a sample to be detected.
The following detailed description of the embodiments of the invention is provided in connection with the accompanying drawings and examples to further illustrate the invention, but should not be construed as limiting the scope of the invention.
Example 1
Preparation of bis (dimethyl pyridinamine) bis (zinc) functionalized detection reagent for detecting N-phosphorylation modified biomolecules
5mmol of 2, 2-dimethylpyridine amine and 5mmol of paraformaldehyde are taken and dissolved in 1mL of mixed solution of isopropanol/water (the isopropanol content is 35%, the pH is 8.0), and the mixture is reacted for half an hour at 80 ℃; 5mmol of N-t-butoxycarbonyl-L-tyrosine was added and reacted at 110℃for 13 hours; purifying by silica gel column chromatography, and dissolving in dichloromethane solution; 10mL of trifluoroacetic acid is added for reaction for 6 hours; adding a methylene dichloride solution containing 5mmol of succinic anhydride, and reacting for 18 hours to obtain the carboxyl-terminated four-arm 2, 2-dimethylpyridine amine.
5mg of carboxyl-terminated four-arm 2, 2-dimethylpyridine amine was weighed and dissolved in 50. Mu.L of dimethyl sulfoxide (DMSO)It was rotated at room temperature for 12 hours to completely dissolve. 10mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was weighed out and dissolved in 80. Mu.L of DMSO. 1mg of N-hydroxysuccinimide (NHS) was weighed and dissolved in 20. Mu.L of DMSO. And adding the two solutions into the completely dissolved carboxyl-terminated four-arm 2, 2-dimethylpyridine amine solution, and performing rotary reaction at room temperature for 12 hours to activate carboxyl to obtain solution A. 2mg of the fourth-generation polyamide-amine type dendrimer was dissolved in 500. Mu.L of absolute methanol to obtain a solution B. Adding 5-50 mu L of solution B into the solution A, fully mixing the solution B and the solution A, and mechanically stirring the mixture at 50 ℃ for reaction for 24 hours to obtain solution C. 20mg Zn (NO) 3 ) 2 ·6H 2 O was dissolved in 30. Mu.L of purified water to give solution D. And adding the solution C into the solution D, and reacting for 24 hours at 50 ℃ in a constant temperature shaking table to obtain the dendritic polymer functionalized by the double zinc functions of the dimedopyrimine. Purifying the obtained dendritic polymer functionalized by the bispyrilamine biszinc functional group by using an ultrafiltration membrane or a dialysis bag (3000 Da), and removing unbound substances to obtain the detection reagent for detecting the N-phosphorylation modified biomolecules functionalized by the bispyrilamine biszinc functional group.
Example 2
Preparation of biotin-modified bis-dimethylpyrazolamide-bis-copper functionalized detection reagent for detecting N-phosphorylation modified biomolecules
5mmol of 2, 2-dimethylpyrazole amine and 5mmol of paraformaldehyde are taken and dissolved in 1mL of isopropanol/water mixed solution (the isopropanol content is 35%, the pH is 8.0), and the mixture is reacted for half an hour at 80 ℃; 5mmol of N-t-butoxycarbonyl-L-tyrosine was added and reacted at 110℃for 13 hours; purifying by silica gel column chromatography, and dissolving in dichloromethane solution; 10mL of trifluoroacetic acid is added for reaction for 6 hours; a dichloromethane solution containing 5mmol of glutaric anhydride is added for reaction for 18 hours to obtain carboxyl-terminated four-arm 2, 2-dimethyl pyrazole amine.
1mg of carboxyl-terminated four-arm 2, 2-dimethylpyrazole amine was dissolved in 50. Mu.L of dimethyl sulfoxide (DMSO), and was allowed to spin at room temperature for 12 hours to complete dissolution. 10mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was weighed out and dissolved in 80. Mu.L of DMSO. 1mg of N-hydroxysuccinimide (NHS) is weighed and dissolved in 20. Mu.L of DMSO. And adding the two solutions into the completely dissolved carboxyl-terminated four-arm 2, 2-dimethylpyrazole amine solution, and performing rotary reaction at room temperature for 12 hours to activate carboxyl to obtain a solution A1. 2mg of the fourth-generation polyamide-amine type dendrimer was dissolved in 500. Mu.L of absolute methanol to obtain a solution B. Adding 5-50 mu L of solution B into the solution A1, fully mixing the solution A and the solution B, and mechanically stirring the mixture at 50 ℃ for reaction for 24 hours to obtain solution C1. Taking 20mg Cu (NO) 3 ) 2 ·6H 2 O was dissolved in 30. Mu.L of purified water to obtain a solution D1. And adding the solution C1 into the solution D1, and reacting for 24 hours at 50 ℃ in a constant-temperature shaking table to obtain the dendrimer functionalized by the di-dimethyl pyrazole amine di-copper functional groups. The resulting dendrimer functionalized with a di-dimethylpyrazolamide di-copper functional group was purified using ultrafiltration membranes or dialysis bags (3000 Da) and then dissolved in 300. Mu.L of phosphate solution to give solution E. 0.8mg of biotin succinimidyl ester was weighed out and dissolved in 300. Mu.L of a phosphate solution to obtain a solution F. And (3) fully mixing the solution E and the solution F, mechanically stirring at room temperature, reacting for 2 hours, purifying by using an ultrafiltration membrane or a dialysis bag (3000 Da), and obtaining the biotin-modified bis-dimethyl-pyrazoloamine bis-copper functionalized detection reagent for detecting the N-phosphorylation modified biomolecules.
Example 3
Preparation of bis-dimethyl pyrimidine amine bis-zinc functionalized detection reagent for detecting N-phosphorylation modified biomolecules
5mmol of 2, 2-dimethylpyrimidinamine and 5mmol of paraformaldehyde are taken and dissolved in 1mL of isopropanol/water mixed solution (the isopropanol content is 35%, the pH is 8.0), and the mixture is reacted for half an hour at 80 ℃; 5mmol of N-t-butoxycarbonyl-L-tyrosine was added and reacted at 110℃for 13 hours; purifying by silica gel column chromatography, and dissolving in dichloromethane solution; 10mL of trifluoroacetic acid is added for reaction for 6 hours; a dichloromethane solution containing 5mmol of glutaric anhydride is added for reaction for 18 hours to obtain carboxyl terminated four-arm 2, 2-dimethyl pyrimidine amine.
5mg of carboxyl-terminated four-arm 2, 2-dimethylpyrimidine amine was weighed and dissolved in 50. Mu.L of dimethyl sulfoxide (DMSO), and rotated at room temperature for 12 hours to complete dissolution. 10mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was weighed and dissolved in 80. Mu.L of DMSOIs a kind of medium. 1mg of N-hydroxysuccinimide (NHS) was weighed and dissolved in 20. Mu.L of DMSO. And adding the two solutions into the completely dissolved carboxyl-terminated four-arm 2, 2-dimethylpyrimidine amine solution, and performing rotary reaction at room temperature for 12 hours to perform carboxyl activation to obtain a solution A2. 2mg of the fourth-generation polyamide-amine type dendrimer was dissolved in 500. Mu.L of absolute methanol to obtain a solution B. Adding 5-50 mu L of solution B into solution A2, fully mixing the two solutions, and mechanically stirring at 50 ℃ for reaction for 24 hours to obtain solution C2. 20mg Zn (NO) 3 ) 2 ·6H 2 O was dissolved in 30. Mu.L of purified water to give solution D. And adding the solution C2 into the solution D, and reacting for 24 hours at 50 ℃ in a constant-temperature shaking table to obtain the dendritic polymer functionalized by the di-dimethyl pyrimidyl amine di-zinc functional group. Purifying the obtained dendritic polymer functionalized by the di-dimethyl pyrimidine amine di-zinc functional group by using an ultrafiltration membrane or a dialysis bag (3000 Da), and removing unbound substances to obtain the detection reagent for detecting the N-phosphorylation modified biomolecules functionalized by the di-dimethyl pyrimidine amine di-zinc.
Example 4
Preparation of detection reagents for detection of N-phosphorylation modified biomolecules based on Di-Zinc-picolinamine functionalization of eighth-generation Polyamide-amine dendrimers
5mmol of 2, 2-dimethylpyridine amine and 5mmol of paraformaldehyde are taken and dissolved in 1mL of mixed solution of isopropanol/water (the isopropanol content is 35%, the pH is 8.0), and the mixture is reacted for half an hour at 80 ℃; 5mmol of N-t-butoxycarbonyl-L-tyrosine was added and reacted at 110℃for 13 hours; purifying by silica gel column chromatography, and dissolving in dichloromethane solution; 10mL of trifluoroacetic acid is added for reaction for 6 hours; a methylene dichloride solution containing 5mmol of glutaric anhydride is added for reaction for 18 hours to obtain carboxyl terminated four-arm 2, 2-dimethyl pyridine amine.
5mg of carboxyl-terminated four-arm 2, 2-dimethylpyridine amine was weighed and dissolved in 50. Mu.L of dimethyl sulfoxide (DMSO), and the mixture was spun at room temperature for 12 hours to dissolve completely. 10mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was weighed out and dissolved in 80. Mu.L of DMSO. 1mg of N-hydroxysuccinimide (NHS) was weighed and dissolved in 20. Mu.L of DMSO. Adding the two solutions into fully dissolved carboxyl-terminated four-arm 2, 2-dimethyl pyridineAnd (3) in the pyrilamine solution, carrying out rotary reaction at room temperature for 12 hours, and activating carboxyl to obtain a solution A. 32mg of the eighth-generation polyamide-amine type dendrimer was dissolved in 500. Mu.L of absolute methanol to obtain a solution B1. Adding 5-50 mu L of solution B1 into the solution A, fully mixing the solution B and the solution A, and mechanically stirring the mixture at 50 ℃ for reaction for 24 hours to obtain solution C3. 20mg Zn (NO) 3 ) 2 ·6H 2 O was dissolved in 30. Mu.L of purified water to give solution D. Adding the solution C3 into the solution D, and reacting for 24 hours at 50 ℃ in a constant temperature shaking table to obtain the double dimethyl pyridine amine double zinc functional group functionalized dendritic polymer based on the eighth generation polyamide-amine dendritic polymer. Purifying the obtained dendrimer based on the bis-dimethyl pyridine amine bis-zinc functional group of the eighth-generation polyamide-amine dendrimer by using an ultrafiltration membrane or a dialysis bag (3000 Da) to remove unbound substances, thereby obtaining the detection reagent for detecting the N-phosphorylation modified biomolecules based on the bis-dimethyl pyridine amine bis-zinc functional group of the eighth-generation polyamide-amine dendrimer.
Example 5
Detection reagent for preparing fluorescein-modified bis-dimethylquinoline amine bis-zinc functionalized detection of N-phosphorylation modified biomolecules
5mmol of 2, 2-dimethylquinoline amine and 5mmol of paraformaldehyde are taken and dissolved in 1mL of isopropanol/water mixed solution (the isopropanol content is 35%, the pH is 8.0), and the mixture is reacted for half an hour at 80 ℃; 5mmol of N-t-butoxycarbonyl-L-tyrosine was added and reacted at 110℃for 13 hours; purifying by silica gel column chromatography, and dissolving in dichloromethane solution; 10mL of trifluoroacetic acid is added for reaction for 6 hours; a dichloromethane solution containing 5mmol of glutaric anhydride is added for reaction for 18 hours to obtain carboxyl terminated four-arm 2, 2-dimethyl quinoline amine.
1mg of carboxyl-terminated four-arm 2, 2-dimethylquinoline amine was weighed and dissolved in 50. Mu.L of dimethyl sulfoxide (DMSO), and the mixture was rotated at room temperature for 12 hours to complete dissolution. 10mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was weighed out and dissolved in 80. Mu.L of DMSO. 1mg of N-hydroxysuccinimide (NHS) was weighed and dissolved in 20. Mu.L of DMSO. Adding the two solutions into the completely dissolved carboxyl-terminated four-arm 2, 2-dimethylquinoline amine solution, and the roomAnd (3) performing warm rotation reaction for 12 hours, and performing carboxyl activation to obtain a solution A3. 2mg of the fourth-generation polyamide-amine type dendrimer was dissolved in 500. Mu.L of absolute methanol to obtain a solution B. Adding 5-50 mu L of solution B into solution A3, fully mixing the two solutions, and mechanically stirring at 50 ℃ for reaction for 24 hours to obtain solution C4. 20mg Zn (NO) 3 ) 2 ·6H 2 O was dissolved in 30. Mu.L of purified water to give solution D. And adding the solution C4 into the solution D, and reacting for 24 hours at 50 ℃ in a constant temperature shaking table to obtain the dendritic polymer functionalized by the bisdimethylquinoline amine and the biszinc functional group.
The obtained dendritic polymer functionalized by the bisdimethylquinoline amine and the zinc functional group is purified by an ultrafiltration membrane or a dialysis bag (3000 Da) and then dissolved in 300 mu L of phosphate solution to obtain a solution E1.
1mg of fluorescein isothiocyanate was dissolved in 300. Mu.L of a phosphate solution to obtain a solution G. After the solution E1 and the solution G are fully mixed, the solution E and the solution G are mechanically stirred at room temperature for reaction for 4 hours, and after purification by a dialysis bag (3000 Da), the detection reagent for detecting N-phosphorylation modified biomolecules by fluorescein modified bis-dimethylquinoline amine bis-zinc functionalization is obtained.
Example 6
Preparation of luminol modified bis-dimethyl pyridinamine bis-zinc functionalized detection reagent for detecting N-phosphorylation modified biomolecules
5mmol of 2, 2-dimethylpyridine amine and 5mmol of paraformaldehyde are taken and dissolved in 1mL of mixed solution of isopropanol/water (the isopropanol content is 35%, the pH is 8.0), and the mixture is reacted for half an hour at 80 ℃; 5mmol of N-t-butoxycarbonyl-L-tyrosine was added and reacted at 110℃for 13 hours; purifying by silica gel column chromatography, and dissolving in dichloromethane solution; 10mL of trifluoroacetic acid is added for reaction for 6 hours; a methylene dichloride solution containing 5mmol of glutaric anhydride is added for reaction for 18 hours to obtain carboxyl terminated four-arm 2, 2-dimethyl pyridine amine.
1mg of carboxyl-terminated four-arm 2, 2-dimethylpyridine amine was weighed and dissolved in 50. Mu.L of dimethyl sulfoxide (DMSO), and the mixture was spun at room temperature for 12 hours to dissolve completely. 10mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was weighed out and dissolved in 80. Mu.L of DMSO. 1mg of N-hydroxysuccinimide (NHS) was weighed out,dissolved in 20. Mu.L DMSO. And adding the two solutions into the completely dissolved carboxyl-terminated four-arm 2, 2-dimethylpyridine amine solution, and performing rotary reaction at room temperature for 12 hours to activate carboxyl to obtain solution A. 2mg of the fourth-generation polyamide-amine type dendrimer was dissolved in 500. Mu.L of absolute methanol to obtain a solution B. Adding 5-50 mu L of solution B into the solution A, fully mixing the solution B and the solution A, and mechanically stirring the mixture at 50 ℃ for reaction for 24 hours to obtain solution C. 20mg Zn (NO) 3 ) 2 ·6H 2 O was dissolved in 30. Mu.L of purified water to give solution D. And adding the solution C into the solution D, and reacting for 24 hours at 50 ℃ in a constant temperature shaking table to obtain the dendritic polymer functionalized by the double zinc functions of the dimedopyrimine.
The obtained dendrimer functionalized with bis (dimethyl pyridine amine) and bis (zinc) functional groups is purified by an ultrafiltration membrane or a dialysis bag (3000 Da) and then dissolved in 300 mu L of phosphate solution to obtain a solution E. 2mL of glutaraldehyde solution containing 25% (W/W) was added, and the mixture was mechanically stirred at room temperature for 2 hours, and purified by ultrafiltration membrane (3000 Da), and the purified product was dissolved in 300. Mu.L of anhydrous methanol to obtain solution H, and 1mg of luminol was weighed and dissolved in 300. Mu.L of anhydrous methanol to obtain solution I. And (3) fully mixing the solution H and the solution I, mechanically stirring at 60 ℃ for reaction for 24 hours, and purifying by using a dialysis bag (3000 Da) to obtain the luminol modified bis-dimethyl pyridine amine bis-zinc functionalized detection reagent for detecting the N-phosphorylation modified biomolecules.
Example 7
The prepared detection reagent for detecting N-phosphorylation modified biomolecules by bis-dimethyl pyridinamine bis-zinc functionalization is applied to spot-printing (Blotting) detection of N-phosphorylation proteins on Nitrocellulose (NC) membrane
After the Nitrocellulose (NC) membrane was placed in the transfer buffer for 1 minute, the membrane was wetted and the water droplets were drained off of the membrane, but not allowed to dry completely. Incubating the membrane by using a water solution of a detection reagent for detecting N-phosphorylation modified biomolecules, wherein the water solution is functionalized by dimetyl pyridinamine dimetyl zinc, taking out the membrane after incubation for 3 hours, completely drying the membrane at room temperature, placing the membrane in 5% (m/v) skimmed milk or 3% (m/v) BSA after drying, sealing the membrane on a shaking table at room temperature for 1 hour, and completely drying the membrane at room temperature to obtain the dimetyl pyridinamine dimetyl zinc functionalized membrane for detecting biomolecules containing phosphorylation modification.
Solutions of different concentrations of N-phosphorylated proteins (N-phosphorylated bovine serum albumin N-BSA and N-phosphorylated beta-Casein N-beta-Casein) and non-N-phosphorylated proteins (bovine serum albumin BSA and beta-Casein) were each taken at 0.5 μl, spotted onto the functionalized membranes prepared above, dried at room temperature for 30 minutes, and after the membranes were completely dried, the membranes were again immersed in a transfer buffer to wet them, and water droplets were drained from the dry membranes. The functionalized membrane loaded with protein spots was blocked with either 5% (m/v) skimmed milk or 3% (m/v) BSA for 1 hour on a room temperature shaker, after which the membrane was washed with HEPES buffer (pH 7.7) (5 min. Times.1). The functionalized membrane carrying the protein spots is diluted with primary antibodies of bovine serum albumin and beta casein respectively according to the recommended dilution ratio of the primary antibodies, and the diluted primary antibodies are incubated on a shaking table at 4 ℃ overnight. After incubation the membrane was washed (10 min. Times.3) with HEPES buffer (pH 7.7). After dilution of the secondary antibody with HEPES buffer (pH 7.7) at the recommended dilution ratio for the secondary antibody, the membrane was incubated for 1 hour on a room temperature shaking table. After incubation the membrane was washed (10 min. Times.3) with HEPES buffer (pH 7.7). Detection was performed with ECL luminophore.
The results are shown in FIG. 3, which shows that the N-phosphorylated protein detection reagent prepared by the method has strong anti-interference capability. The reagent can realize the selective detection of the N-phosphorylated protein under the interference of high concentration non-N-phosphorylated protein (100 times) by combining antibodies capable of specifically recognizing the protein (the antibodies used in the reagent are conventional commercial antibodies capable of specifically recognizing bovine serum albumin and casein and are not special antibodies capable of recognizing N-phosphorylated sites), and the minimum detection sensitivity of the reagent to the N-phosphorylated protein can reach 1ng.
Example 8
The prepared biotin-modified bis-dimethylpyrazolamide bis-zinc functionalized detection reagent for detecting N-phosphorylation modified biomolecules is applied to spot imprinting (Blotting) detection of N-phosphorylation proteins on polyvinylidene fluoride (PVDF) membranes.
Polyvinylidene fluoride (PVDF) film was activated with methanol and then dried. Solutions of different concentrations of N-phosphorylated protein (N-phosphorylated bovine serum albumin, N-BSA) and non-N-phosphorylated protein (bovine serum albumin, BSA) were each taken at 0.5. Mu.L and spotted onto activated and air-dried PVDF membranes. The membrane was then placed in blocking solution (0.25 mol/L ammonium acetate, 0.5mol/L sodium chloride, 0.5% Tween 20, 5% BSA) and incubated on a shaker for 1 hour. The mixture was washed twice with three distilled water for 5 minutes. The membrane was placed in 10mL of pH 8.8 ammonium acetate (0.25 mol/L) buffer containing 20. Mu.L of the detection reagent of the present invention, 5% BSA, 0.5% Tween 20, and incubated on a shaker for 1 hour. Washing with pH 8.8 buffer (containing 0.25mol/L ammonium acetate, 0.5mol/L sodium chloride, 0.5% Tween 20) for 4 times each for 5 minutes; the mixture was washed with ammonium acetate (0.05 mol/L) at pH 8.8 for 5 minutes. The membrane was placed in an Avidin-HRP ammonium acetate solution (pH 8.8,0.25mol/L ammonium acetate, 0.5mol/L sodium chloride, 0.5% Tween 20, 5% BSA) with a 1:8000 dilution and incubated for 45 min. The cells were washed with pH 8.8 buffer (containing 0.25mol/L ammonium acetate, 0.5mol/L sodium chloride, 0.5% Tween 20) for 4 times, each time for 5 minutes. The results of the detection with ECL luminescence are shown in fig. 4.
According to FIG. 4, the N-phosphorylated protein detection reagent prepared by the invention can quantitatively detect N-phosphorylated protein within the range of 1-50 ng, has good selectivity to N-phosphorylated protein, has a quantitative detection range of more than 2 orders of magnitude for N-phosphorylated protein, has good linear relationship, and has R 2 Is more than 0.95 percent; the gray value of the N-phosphorylated protein is linearly and positively correlated with the spotting amount of the N-phosphorylated protein through regression analysis. The results show that the N-phosphorylated protein detection reagent prepared by the method has quantitative detection capability on the N-phosphorylated protein.
Example 9
The prepared fluorescein-modified bis (dimethylquinoline) amine bis (zinc) functionalized detection reagent for detecting N-phosphorylation modified biomolecules is applied to spot-print detection of N-phosphorylation proteins on a polyvinylidene fluoride (PVDF) membrane.
The procedure and flow used are very similar to example 7. Except that the membrane was incubated in a solution containing the detection reagent of the present invention and rinsed, and then directly detected by a fluorescence imager.
Example 10
Example 1 application of bis-dimethyl-pyridinamine bis-zinc functionalized detection reagents for detection of N-phosphorylation modified biomolecules to dynamic real-time immunoblotting (Western Blotting) detection of N-phosphorylation levels of N-phosphorylated proteins on polyvinylidene fluoride (PVDF) membranes the procedure and flow are similar to example 7. The results are shown in FIG. 5. As can be seen from FIG. 5, the detection reagent of the present invention can be used for the real-time dynamic monitoring of the change of the N-phosphorylation modification level of the N-phosphorylated protein.
Example 11
The procedure and flow for the use of the biotin-modified bis-dimethylpyrazolamide-bis-copper functionalized detection reagents for detecting N-phosphorylated biomolecules prepared in example 2 are similar to example 8, and the results of the reusability and stability evaluation when applied to immunoblotting (Western Blotting) detection of N-phosphorylated proteins on polyvinylidene fluoride (PVDF) membranes are shown in FIG. 6. As can be seen from FIG. 6, the detection reagent of the present invention can be repeatedly applied to the selective detection of N-phosphorylated proteins, and the long-term storage has no significant effect on the detection effect of the detection reagent of the present invention.
Example 12
The prepared biotin-modified bis-dimethylpyrazolamide bis-zinc functionalized detection reagent for detecting N-phosphorylation modified biomolecules is applied to detection (ELISA) of N-phosphorylated proteins in a microplate.
N-phosphorylated protein was diluted to a concentration of between 10ng/mL and 1mg/mL with 0.05mol/L of a carbonic acid buffer pH 9.5, and 0.1mL of the solution was added to each reaction well of the polystyrene plate at 4℃overnight. The next day, the solution in the wells was discarded and washed 3 times with pH 9.0 ammonium acetate buffer for 3 minutes each. To each reaction well, 100. Mu. LpH 9.0.0 ammonium acetate (0.25 mol/L) buffer containing 0.2. Mu.L of the detection reagent of the present invention, 1% BSA, and 0.5% Tween 20 was added, and incubated at 37℃for 0.5 to 1 hour, and washed 3 times with pH 9.0 ammonium acetate buffer for 3 minutes each. To each well was added a 1:3000 dilution of Avidin-HRP ammonium acetate solution (pH 8.8,0.25mol/L ammonium acetate, 0.5mol/L sodium chloride, 0.5% Tween 20, 5% BSA) and incubated at 37℃for 45 minutes. The cells were washed with pH 8.8 buffer (containing 0.25mol/L ammonium acetate, 0.5mol/L sodium chloride, 0.5% Tween 20) for 5 minutes for 4 times. Detection was performed with ECL luminophore. The results are shown in FIG. 7.
As can be seen from FIG. 7, the detection reagent of the present invention can realize quantitative analysis of N-phosphorylation level in biological sample, has good selectivity to N-phosphorylated protein, quantitative detection range of N-phosphorylated protein is more than 2 orders of magnitude, and has good linear relationship, R 2 Is 0.95 or more.
Example 13
The prepared fluorescein-modified bis (dimethylquinoline) amine bis (zinc) functionalized detection reagent for detecting N-phosphorylation modified biomolecules is applied to detection (ELISA) of N-phosphorylation proteins in a microplate.
The procedure and flow used are very similar to that of example 12. Except that the well plate was incubated in a solution containing the detection reagent of the present invention and rinsed, and then detected by a fluorogenic microplate reader.
As can be seen from the above examples, the detection reagent provided by the present invention can be directly used for detecting N-phosphorylation modified biomolecules, and on the one hand, quantitative analysis of the overall N-phosphorylation modification level of all N-phosphorylation modified biomolecules in a sample to be detected can be realized; on the other hand, the kit can be combined with a conventional reagent capable of realizing the specific detection of the biomolecules to realize the high-sensitivity and high-specificity detection of the N-phosphorylation modified biomolecules, can be used for detecting more than 1ng of biomolecules containing the N-phosphorylation modification, has strong anti-interference capability, can selectively detect the biomolecules containing the N-phosphorylation modification under 100 times of the interference of the biomolecules without the N-phosphorylation modification, and has good stability.
Although the foregoing embodiments have been described in some, but not all embodiments of the invention, other embodiments may be obtained according to the present embodiments without departing from the scope of the invention.
Claims (10)
1. A detection reagent for an N-phosphorylated modified biomolecule, comprising a bimetallic functionalized dendrimer of the Pincer type; the functionalized dendritic polymer is prepared from raw materials comprising an A component and a B component;
the A component is a dendritic polymer;
the component B is a piver type ligand-bimetallic coordination compound; the Picer type ligand-bimetallic coordination compound is a Picer type coordination-double zinc coordination compound or a Picer type coordination-double copper coordination compound.
2. The detection reagent according to claim 1, wherein the functionalized dendrimer is further bonded with a C component that is a functional group that can achieve direct detection or an antibody group that can achieve direct detection.
3. The detection reagent of claim 1, wherein the functionalized dendrimer is further bound with a D component that is a functional group that can bind to a C component or an antibody group that can bind to a C component.
4. The detection reagent according to claim 1, wherein the dendrimer is a polyamide-amine dendrimer or a co-embedded dendrimer of a polyamide-amine dendrimer.
5. The detection reagent according to claim 4, wherein the polyamide-amine type dendrimer is one or more of second to tenth generations.
6. The reagent according to claim 1, wherein the component B is a piter type ligand-bimetallic complex containing a heteroatom ligand, which is obtained from a piter type ligand containing a heteroatom ligand and a metal ion;
the heteroatom comprises one or more of oxygen, nitrogen and sulfur;
the Picer type ligand containing heteroatom coordination comprises one or more of 2,2' -dimethylpyridine amine, 2' -dimethylpyrazole amine, 2' -dimethylpyrimidine amine, 2' -dimethylquinoline amine, 2' -dimethylthiazole amine, 2' -dimethylfuran amine and 2,2' -dimethylisoquinoline amine.
7. A detection reagent according to claim 2 or 3, wherein the functional group in the C-component which enables direct detection comprises a fluorescent group or a luminescent group;
The functional group which can be combined with the C component in the D component comprises biotin, hapten or crosslinking group.
8. The method for producing a detection reagent according to any one of claims 1 to 7, comprising the steps of:
(1) Mixing the dendritic polymer solution with a Picer type ligand solution and a metal ion solution for a coupling reaction to obtain a solution containing a Picer type bimetal functionalized dendritic polymer; the metal ion solution is zinc ion solution or copper ion solution;
(2) Purifying the solution containing the Pincer type bimetal functionalized dendritic polymer to obtain the detection reagent of the N-phosphorylation modified biological molecule.
9. The preparation method according to claim 8, wherein the preparation method of the Pincer type ligand solution comprises the following steps: mixing an amine compound, paraformaldehyde and a first solvent for a Mannich reaction; carrying out first amidation reaction on the obtained reaction product and N-t-butoxycarbonyl-L-tyrosine; purifying the obtained product, and then mixing the purified product with dichloromethane and trifluoroacetic acid to carry out a protective group removal reaction; carrying out a second amidation reaction on the obtained reaction product and an anhydride-containing dichloromethane solution to obtain a carboxyl-terminated four-arm amine compound; and mixing the carboxyl-terminated four-arm amine compound, dimethyl sulfoxide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide for performing a third amidation reaction to obtain a Picer type ligand solution.
10. Use of a detection reagent according to any one of claims 1 to 7 or a detection reagent obtained by a method according to any one of claims 8 to 9 in the preparation of a kit for detecting a biological molecule comprising an N-phosphorylation modification.
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