CN106496589A - A kind of both sexes carboxylic acid 3-dimensional metal coordination polymer and its preparation method and application - Google Patents

A kind of both sexes carboxylic acid 3-dimensional metal coordination polymer and its preparation method and application Download PDF

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CN106496589A
CN106496589A CN201611077693.XA CN201611077693A CN106496589A CN 106496589 A CN106496589 A CN 106496589A CN 201611077693 A CN201611077693 A CN 201611077693A CN 106496589 A CN106496589 A CN 106496589A
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CN106496589B (en
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谢宝平
陈金香
孙斌
杨水平
邱桂华
张茉莉
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Southern Medical University
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Abstract

The present invention relates to a kind of both sexes carboxylic acid 3-dimensional metal coordination polymer and its preparation method and application, specifically disclose a kind of soluble amphoteric carboxylic acid 3-dimensional metal coordination polymer, the main part of three-dimensional copper metal coordination polymer is N (3,5 dicarboxyl benzyls) (3 carboxyl) pyridinium bromide, assistant ligand is 1,2 two (4 pyridine radicals) ethene, central atom is copper.Product raw material sources of the present invention are extensive, be readily available, synthesis technique is simple, reaction condition is gentle, reproducible.Both sexes carboxylic acid three-dimensional copper metal coordination polymer of the present invention has good water solubility, the detection of the nucleotide sequences such as AIDS, Sudan virus and dengue fever virus can be used successfully to, and have the advantages that fast and convenient, efficient and sensible, have a good application prospect in the detection of relevant disease.

Description

A kind of both sexes carboxylic acid 3-dimensional metal coordination polymer and its preparation method and application
Technical field
The present invention relates to technical field of biological material, and in particular to a kind of preparation of both sexes carboxylic acid three-dimensional copper metal coordination polymer Method and application.
Background technology
Detect that nucleic acid molecules have become scientific researcher concern according to fluorescence spectroscopy principle application fluorescence probe in recent years A big focus, this method has the advantages that sensitive, quick, easy and inexpensive.According to the literature, carbon nanomaterial, Nano metal, part metals coordination polymer can be used as detection of nucleic acids fluorescence probes.But the materials such as carbon nanomaterial and nano metal Material because the complicated loaded down with trivial details, production cost of synthesis is high, the shortcomings of be insoluble in water, limit these materials in terms of detection of nucleic acids should With research.
Metal coordinating polymer is that a class is combined by co-ordinate covalent bond with organic ligand by metal ion, and passes through from group The novel porous polymeric material that the mode of dress is obtained.Metal coordinating polymer has specific surface area big, and hole density is high, hole is big Little people have applied it to biological medicine related field the advantages of can adjust, be easy to functionalization, such as carry medicine, biology and make Shadow, to detection of biological micromolecule and macromolecular etc..Metal coordinating polymer has one-dimensional, the structure of two and three dimensions, wherein There is the metal coordinating polymer of three-dimensional pore space structure because of its larger porosity, there is to probe nucleic acid molecule preferably absorption Effect, so that be more expected to become excellent viral nucleic acid molecule probe.
Although there is the metal coordinating polymer with three-dimensional pore space structure lot of advantages, part rigid ligand to construct Metal coordinating polymer poorly water-soluble, is susceptible to pore space structure after meeting water and caves in, and seriously hinders its nucleic acid molecules detection work( The real application research of energy.Therefore, the hydrostable metal coordinating polymer material of design synthesis is further to further investigate which The key of nucleic acid molecular probe function.Quaternary ammonium salt both sexes carboxylic acid is the larger part of a class polarity, and research is found with such part The metal coordinating polymer built with metal ion self assembly not only has abundant skeleton structure, also stable with good water Property, therefore the 3-dimensional metal polycomplexation with good water stability can be built by designing synthesis quaternary ammonium salt both sexes Carboxylic acid ligand Compound is applied to the detection of nucleic acid molecules.
Content of the invention
One of technical assignment of the present invention is to make up the deficiencies in the prior art, there is provided a kind of both sexes carboxylic acid three-dimensional copper gold The preparation method of category coordination polymer, the i.e. preparation method of [Cu (Dcbcp) is (bpe)] metal coordinating polymer material, the method Raw materials used wide material sources, be readily available, synthesis technique is simple, reaction condition is gentle, reproducible.
The two of the technical assignment of the present invention are to provide the purposes of the both sexes carboxylic acid three-dimensional copper metal coordination polymer, the material There is good aqueous solubility and water stability, and effective inspection of associated nucleic acid sequences can be used successfully to as detection of nucleic acids fluorescence probe Survey, and have the advantages that fast and convenient, efficient and sensible, before there is applications well in the nucleic acid series detection related to disease Scape.
Technical proposal that the invention solves the above-mentioned problems is as follows：
1st, a kind of preparation of both sexes carboxylic acid three-dimensional copper metal coordination polymer
The both sexes carboxylic acid three-dimensional copper metal coordination polymer is both sexes tricarboxylic acids part and the common structure of bipyridyl assistant ligand Build and obtain, it is characterised in that main part is N- (3,5- dicarboxyl benzyl)-(3- carboxyls)-pyridinium bromide (H3DcbcpBr), aid in Part is 1,2-di(4-pyridyl)ethylene (bpe), and its ligand molecular structure is as shown in Figure 1.The element of the copper coordination polymer Analysis mode is C27H19N3O6Cu·H2O, calculated value are C 57.54, H 3.73, N 7.46；Experiment value is C 57.42, H3.76, N,7.28；Ir data (KBr) is：3546(s),3038(m),1604(s),1436(s),1162(m),1067(m), 983(m),816(m),746(m),718(m),671(m),559(m),457(m).
The single crystal diffraction of the both sexes carboxylic acid three-dimensional copper metal coordination polymer enters line number using Bruker APEX II diffractometers According to collection, the cell parameter drawn through crystal parsing is respectively： α=90 °, β=121.969 (4) °, γ=90 °.Other related crystallographic parameters are listed in subordinate list 1, and important bond distance's bond angle is shown in Subordinate list 2.
[Cu (Dcbcp) is (bpe) for table 1n] crystallographic parameter
aR=| | Fo|-|Fc|/|Fo|.bWR={ w (Fo 2-Fc 2)2/w(Fo 2)2}1/2.cGOF={ w ((Fo 2-Fc 2)2)/ (n-p)}1/2, where n=number of reflections and p=total numbers of parameters refined
[Cu (Dcbcp) is (bpe) for table 2n] part bond distanceWith bond angle [°]
Symmetrytransformations used to generate equivalent atoms:#1:x,-y,z- 1/2；#2:
x+1/2,-y+1/2,z+1/2；
#3:-x+1/2,y+1/2,-z+1/2；#4:x,-y,z+1/2；#5:-x+1/2,y-1/2,-z+1/2；#6:x-1/ 2,-y+1/2,z-1/2
X-ray single crystal diffraction result shows that the copper metal coordination polymer belongs to monoclinic system, and space group is C2/c, per Individual asymmetric cell contains [Cu (Dcbcp) is (bpe)] molecule, the center of Ni metal (II) and the N from two bpe molecules Four O atom coordinations on three carboxyls on atom and three Dcbcp parts.Two of which carboxyl belongs to monodentate ligand mould Formula, also one carboxyl belong to chelating coordination modes, are consequently formed octahedral coordination configuration.Each two Cu atom is by a pair of μ COO connects, and Cu atoms further form [Cu with carboxyl chelating ligands2(Dcbcp)4] secondary structure, [a Cu2 (Dcbcp)4] six identical structures of secondary structure and surrounding are connected, and form the planar structure of two dimension on bc axial planes.Cu atoms Combination is coordinated on a direction of principal axis with two bpe molecules further, is ultimately formed the structure of three-dimensional, is seen Fig. 2.
Both sexes copper carboxylate metal coordinating polymer of the present invention adopts preparation method commonly used in the art, e.g., by N- The reaction of (3,5- dicarboxyl benzyls)-(3- carboxyls)-pyridinium bromide, bis- (4- pyridine radicals) ethene of 1,2- and mantoquita is obtained.The present invention The method that people recommends, comprises the steps of：
(1) N- (3,5- dicarboxyl benzyl)-(3- carboxyls)-pyridinium bromide is scattered in distilled water, molten with 0.1M NaOH Liquid adjusts pH to 7.0, obtains main ligand solution
(2) water will be dissolved in the copper sulphate of N- (3,5- dicarboxyl benzyls)-(3- carboxyls)-pyridinium bromide equimolar amounts In, obtain copper salt solution；
(3) will be with the 1,2- bis- (4- pyridine radicals) of N- (3,5- dicarboxyl benzyls)-(3- carboxyls)-pyridinium bromide equimolar amounts Ethylene dissolution obtains assistant ligand solution in DMF；
(4) copper salt solution that step (2) is obtained is added to step (1) to obtain, in main ligand solution, reaction being stirred at room temperature Filter after 0.5h, the assistant ligand solution that step (3) is obtained is added in filtrate, is filtered after stirring 0.5h, filtrate packing yuxin dimension Your bottle Program is warming up to 100 DEG C of reaction 72h, and programmed cooling separates out green lump shaped crystalline to 30 DEG C again afterwards, collects crystal, second Ether is vacuum dried after rinsing, and obtains described both sexes copper carboxylate metal coordinating polymer [Cu (Dcbcp) is (bpe)].
Synthesized both sexes copper carboxylate metal coordinating polymer [Cu (Dcbcp) is (bpe)] is soaked in 48h in water, is taken out Tested by powder diffraction afterwards, compared with unsoaked, XRD is basically identical, its result is shown in accompanying drawing 3, the copper metal is described Coordination polymer skeleton does not cave in, and there is good stability to water.Under the conditions of 25 DEG C, the copper metal coordination polymer is in water In solubility up to 0.6mg/mL.
2nd, the both sexes carboxylic acid three-dimensional copper metal coordination polymer that prepared by method as defined above, detects nucleic acid for fluorescence probe Molecule
Cleaning Principle is as follows：Copper metal coordination polymer is first by electrostatic, pi-pi accumulation and (or) hydrogen bond action and P- DNA (DNA with fluorescence labeling material) combines to form compound system and produces fluorescent quenching phenomenon.Add and P- in system The targeting DNA/RNA (Target DNA/RNA) of DNA base complementary relationship, because very strong between Target DNA/RNA and P-DNA Complementary pairing effect, make P-DNA depart from quencher material and Target DNA/RNA and combine to form double-strand or heteroduplex DNA, Because the interaction of double-strand or heteroduplex DNA and MOF is weaker, cause so as to depart from from copper metal coordination polymer material surface Fluorescence is recovered, the purpose for reaching detection DNA/RNA by fluorescence spectrum characterization fluorescent quenching and recovery degree.
Concrete detecting step is as follows：
(1) compound on fluorescence marks the fluorescent quenching of P-DNA
100 μM of P-DNA solution is diluted to 50nM with 100 μM of Tris-HCl cushioning liquid, as liquid to be titrated；Will Compound be dissolved in 100 μM of Tris-HCl cushioning liquid be made into 500 μM (P-DNA containing 50nM) solution be titrating solution.Take P- Titrating solution in fluorescence cuvette is pressed volume gradient and is added in cuvette, record system fluorescence intensity by DNA liquid 2mL to be titrated Change (λex=480nm) until fluorescent quenching reaches saturation, calculate fluorescent quenching efficiency QE%.
QE%=(1-FM/F0(the F of) × 100%0：P-DNA initial fluorescent intensities；FM：The fluorescence being quenched when reaching saturation is strong Degree)
(2) compound system is in the recognition detection to Target DNA/RNA sequences
The determination in reaction time：On above-mentioned experiment basis, by dilution after Target DNA/RNA solution to be checked minimum Volume (10 μ L) is added in the compound system that fluorescent quenching reaches saturation, makes Target DNA/RNA reach minimum in system Concentration 25nM, monitoring fluorescence intensity over time, determine that the recovery of system fluorescence reaches the time of maximum as detection The reaction time R of Target DNA/RNAT.
The measure of fluorescence anabiosis rate：Detect the reaction time of Cmin Target DNA/RNA as mark with compound system Standard, record compound system fluorescence intensity increase and the extent of recovery with Target DNA/RNA concentration, until fluorescence recovery reaches Saturation, calculates fluorescence recovery efficiency RE.
RE=FT/FM- 1 (FT：Recovery reaches fluorescence intensity during saturation；FM：The fluorescence intensity being quenched when reaching saturation)
The calculating of test limit：The test limit (LOD) that compound system is determined is calculated to Target DNA/RNA with following formula.
LOD=3 σ/S (σ：For not adding the fluorescence intensity standard deviation before Target DNA/RNA, S is multiple for correction fluorescence Soviet Union's slope of curve)
(3) recognition detection of the copper metal coordination polymer material of the present invention to viral associated nucleic acid sequences
Copper metal coordination polymer material of the present invention is used for AIDS virus, Sudan virus, dengue virus related The detection of nucleic acid, acts on good detection, compared with other metal coordinating polymer material tests nucleic acid of document report, Copper metal coordination polymer of the present invention is used for HIV-1ds-DNA, SUDV RNA and DENV-2RNA detection and there is detection time The low advantage of shorter, test limit, which the results are shown in Table 3.
Metal coordinating polymer detection performance delivered by 3 copper metal coordination polymer of the present invention of table and part document
Beneficial effect
1) product raw material sources of the present invention extensively, be readily available.
2) Product formation process is simple of the present invention, reaction condition are gentle, can recrystallize acquisition, nontoxic nothing in aqueous Evil, without the need for inert gas shielding, has accomplished green chemical synthesis, and yield is up to more than 75%.
3) product of the present invention has Detection results to HIV-1ds-DNA, SUDV RNA and DENV-2RNA；The quick letter of detection Just, efficient and sensible, has a good application prospect in the nucleic acid series detection of the relevant disease that virus causes.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all spirit in the present invention and Within principle, any modification, equivalent substitution and improvements that is made etc. should be included within the scope of the present invention.
Description of the drawings
Fig. 1 is the ligand structure of both sexes carboxylic acid three-dimensional copper metal coordination polymer of the present invention；
Fig. 2 is the crystal structure of both sexes carboxylic acid three-dimensional copper metal coordination polymer of the present invention；
Fig. 3 is the powder diagram of both sexes carboxylic acid three-dimensional copper metal coordination polymer of the present invention；
Fig. 4 be both sexes carboxylic acid of the present invention three-dimensional copper metal coordination polymer to P1- DNA fluorescent quenching curves；
Fig. 5 is T in compound system1- DNA fluorescence recoveries intensity with concentration curve (a) and changes over curve (b)；
Fig. 6 be both sexes carboxylic acid of the present invention three-dimensional copper metal coordination polymer to P2- DNA fluorescent quenching curves；
Fig. 7 is T in compound system2- RNA fluorescence recoveries intensity with concentration curve (a) and changes over curve (b)；
Fig. 8 be both sexes carboxylic acid of the present invention three-dimensional copper metal coordination polymer to P3- DNA fluorescent quenching curves；
Fig. 9 is T in compound system3- RNA fluorescence recoveries intensity with concentration curve (a) and changes over curve (b).
Specific embodiment
Embodiment 1 prepares metal coordinating polymer
1st, the preparation of both sexes carboxylic acid three-dimensional copper metal coordination polymer
(1) N- (3,5- dicarboxyl benzyls)-(3- carboxyls)-pyridinium bromide (0.764g, 2mmol) is scattered in distilled water (70mL), in, adjust pH to 7.0 with 0.1M NaOH solutions, obtain main ligand solution；
(2) by CuSO4·5H2O (0.391g, 2mmol) is dissolved in distilled water (20mL), obtains copper salt solution；
(3) 1,2-di(4-pyridyl)ethylene (0.360g, 2mmol) is dissolved in DMF solution (5mL), is aided in Ligand solution；
(4) copper salt solution that step 2 is obtained is added dropwise in the main ligand solution that step 1 is obtained, is stirred at room temperature anti- Filter after answering 0.5h, obtain settled solution；
(5) the assistant ligand solution for obtaining step 3 is added in the settled solution that step 4 is obtained, mistake after stirring 0.5h Filter obtains blue settled solution.Filtrate is dispensed in yuxin Wei Er bottles carries out temperature programming (1h, 25 DEG C to 100 DEG C), in 100 DEG C Reaction 72h, carries out programmed cooling (48h, 100 DEG C to 30 DEG C) afterwards again and separates out green lump shaped crystalline, collect crystal, washed with ether After be vacuum dried, obtain described both sexes carboxylic acid three-dimensional copper metal coordination polymer [Cu (Dcbcp) is (bpe)] (through calculating yield For 85%).
2nd, the identification of both sexes carboxylic acid three-dimensional copper metal coordination polymer [Cu (Dcbcp) is (bpe)]
It is green bulk crystals under the both sexes carboxylic acid three-dimensional copper metal coordination polymer normal temperature, molecular formula is [Cu (Dcbcp) (bpe)], the Dcbcp in formula is N- (3,5- dicarboxyl benzyl)-(3- carboxyls)-pyridinium bromide, and bpe is 1,2- bis- (4- pyridine radicals) ethene.Analyzed and identified by elementary analysis, IR spectrum and X-ray single crystal diffraction, as a result as follows.
(1) elementary analysis C27H19N3O6Cu·H2O, calculated value:C 57.54,H 3.73,N 7.46；Experiment value:C 57.42,H3.76,N,7.28.
(2) infrared spectrum (KBr disc, cm-1)ν3546(s),3038(m),1604(s),1436(s),1162(m), 1067(m),983(m),816(m),746(m),718(m),671(m),559(m),457(m).
(3) related crystallographic parameters：Single crystal diffraction carries out Data Collection using Bruker APEX II diffractometers, through crystalline substance The cell parameter that body parsing draws is respectively：α= 90 ° ,=121.969 (4) °, γ=90 °.Other related crystallographic parameters are listed in subordinate list 1, and important bond distance's bond angle sees attached list 2. X-ray single crystal diffraction result shows that the both sexes carboxylic acid three-dimensional copper metal coordination polymer belongs to monoclinic system, and space group is C2/c, each asymmetric cell contain [Cu (Dcbcp) is (bpe)] molecule, the center of Ni metal (II) with from two bpe Four O atom coordinations on three carboxyls on the N atoms of molecule and three Dcbcp parts.Two of which carboxyl belongs to monodentate Coordination mode, also one carboxyl belong to chelating coordination modes, are consequently formed octahedral coordination configuration.Each two Cu atom by A pair of μ COO connections, Cu atoms further form [Cu with carboxyl chelating ligands2(Dcbcp)4] secondary structure, [a Cu2 (Dcbcp)4] six identical structures of secondary structure and surrounding are connected, and form the planar structure of two dimension on bc axial planes.Cu atoms Combination is coordinated on a direction of principal axis with two bpe molecules further, ultimately forms the structure of three-dimensional.
(4) water stability：Synthesized [Cu (Dcbcp) is (bpe)] is soaked in water after 48h carries out powder diffraction survey Examination, and compare with unsoaked, it is as a result XRD before and after immersion basically identical (see accompanying drawing 3), the copper metal polycomplexation is described Polymer backbone does not cave in, and there is good stability to water.
(5) solubility in water：Under the conditions of 25 DEG C, the solubility of the copper metal coordination polymer in water is 0.6mg/ mL.
Embodiment 2 is with 1 copper metal coordination polymer of embodiment to AIDS virus nucleic acid Sequence Detection
(1) determination of AIDS virus HIV-1 nucleotide sequences to be checked
By GeneBank and the retrieval-by-unification of other Relational databases, filter out in AIDS virus HIV-1 conserved sequences 20-30bp sizes, have 100% specific ds DNA sequence dnas, artificial synthesized as sequence to be checked, i.e. target DNA, and Fluorescence labeling (P-DNA) is carried out to its complementary strand cDNA sequence, sequence is as follows：
Target1DNA sequence dna (T1-DNA)：5'-AGAAAAAAGAAGAA-3'
P1- DNA sequence dna complementary strand (P1-DNA)：5'-FAM-TTCTTCTTTTTTCT-3'
(2) the copper metal coordination polymer aqueous solution is prepared
Copper metal coordination polymer is dissolved in distilled water and is configured to solution that concentration is 250mM and with sterilizing filter pair Solution carries out degerming, and sealing preserve is stand-by as stock solution.
(3) metal coordinating polymer is to fluorescence labeling P1The fluorescent quenching of-DNA
With 100 μM of Tris-HCl cushioning liquid by 100 μM of P1- DNA solution is diluted to 50nM, used as liquid to be titrated；Take Metal coordinating polymer stock solution is diluted to 500 μM of (P containing 50nM with 100 μM of Tris-HCl cushioning liquid1- DNA) as drop Determine liquid.Take P1Titrating solution in fluorescence cuvette is pressed volume gradient and is added in cuvette, record body by-DNA liquid 2mL to be titrated It is the change (λ of fluorescence intensityex=480nm) until fluorescent quenching reaches saturation, calculate fluorescent quenching efficiency QE%.The copper metal Coordination polymer is to fluorescently-labeled P1The fluorescent quenching rate of-DNA is 65.0%.
(4) compound system is to T to be checked1The recognition detection of-DNA sequence dna
On above-mentioned experiment basis, by dilution after T to be checked1- DNA solution (10 μ L) is added to fluorescent quenching and reaches saturation Compound system in, make T1- DNA reaches Cmin 25nM in system, and monitoring fluorescence intensity over time, determines body It is that fluorescence recovery reaches the time of maximum as detection T1The reaction time R of-DNAT.T is detected with compound system1- DNA's is anti- R between seasonableTFor standard, record compound system fluorescence intensity is with T1- DNA concentration increases and the extent of recovery, until fluorescence recovery reaches To saturation, fluorescence anabiosis rate R is calculatedEAnd test limit.Copper metal coordination polymer and fluorescently-labeled P1- DNA forms compound system It is 23min, fluorescence anabiosis rate R to the detection reaction time of specific ds DNA sequence dnas in AIDS virus HIV-1 conserved sequencesE For 0.98, detection is limited to 81pM.The result shows both sexes carboxylic acid three-dimensional copper metal coordination polymer of the present invention to AIDS The nucleotide sequence of viral HIV-1 has preferable detection effect.
Embodiment 3 is detected to Sudan virus nucleotide sequence with 1 copper metal coordination polymer of embodiment
(1) determination of Sudan virus SUDV nucleotide sequences to be checked
By GeneBank and the retrieval-by-unification of other Relational databases, filter out in Sudan virus SUDV conserved sequences 20-30bp sizes, have 100% specific RNA sequence, artificial synthesized as sequence to be checked, i.e. target RNA.Consider The complementary strand of target RNA is replaced as its complementary DNA (cDNA), and cDNA sequence is carried out by the relative unstability of RNA Fluorescence labeling, i.e. probe DNA, sequence are as follows：
Target2RNA sequence (T2-RNA)：5'-GAUGAGGACAAACUUUUUAA-3'
P2- DNA sequence dna complementary strand (P2-DNA)：5'-FAM-TTAAAAAGTTTGTCCTCATC-3'
(2) the copper metal coordination polymer aqueous solution is prepared
Copper metal coordination polymer is dissolved in distilled water and is configured to solution that concentration is 250mM and with sterilizing filter pair Solution carries out degerming, and sealing preserve is stand-by as stock solution.
(3) metal coordinating polymer is to fluorescence labeling P2The fluorescent quenching of-DNA
With 100 μM of Tris-HCl cushioning liquid by 100 μM of P2- DNA solution is diluted to 50nM, used as liquid to be titrated；Will Take metal coordinating polymer stock solution 500 μM of (P containing 50nM are diluted to 100 μM of Tris-HCl cushioning liquid2- DNA) conduct Titrating solution.Take P2Titrating solution in fluorescence cuvette is pressed volume gradient and is added in cuvette by-DNA liquid 2mL to be titrated, record Change (the λ of system fluorescence intensityex=480nm) until fluorescent quenching reaches saturation, calculate fluorescent quenching efficiency QE%.The copper gold Category coordination polymer is to fluorescently-labeled P1The fluorescent quenching rate of-DNA is 83.0%.
(4) compound system is to T to be checked2The recognition detection of-RNA sequence
On above-mentioned experiment basis, by dilution after T to be checked2- RNA solution (10 μ L) is added to fluorescent quenching and reaches saturation Compound system in, make T2- RNA reaches Cmin 25nM in system, and monitoring fluorescence intensity over time, determines body It is that fluorescence recovery reaches the time of maximum as detection T2The reaction time R of-RNAT.T is detected with compound system2- RNA's is anti- R between seasonableTFor standard, record compound system fluorescence intensity is with T2- RNA concentration increases and the extent of recovery, until fluorescence recovery reaches To saturation, fluorescence anabiosis rate R is calculatedEAnd test limit.Copper metal coordination polymer and fluorescently-labeled P2- DNA forms compound system It is 23min, fluorescence anabiosis rate R to the Sudan virus SUDV RNA sequences detection reaction timeEFor 2.4, detection is limited to 31pM.The knot Fruit shows that both sexes carboxylic acid three-dimensional copper metal coordination polymer of the present invention has preferably inspection to Sudan virus nucleotide sequence Survey is acted on.
Embodiment 3 is detected to 2 type nucleic acid sequence of dengue fever with 1 copper metal coordination polymer of embodiment
(1) determination of 2 type of dengue fever virus DENV-2 nucleotide sequences to be checked
By GeneBank and the retrieval-by-unification of other Relational databases, filter out 2 type of dengue fever virus DENV-2 and guard 20-30bp sizes in sequence, there is 100% specific RNA sequence, artificial synthesized as sequence to be checked, i.e. target RNA. The relative unstability of RNA is considered, the complementary strand of target RNA is replaced as its complementary DNA (cDNA), and to cDNA sequences Row carry out fluorescence labeling, i.e. probe DNA, and sequence is as follows：
Target3RNA sequence (T3-RNA)：5'-UGGUGCUGUUGAAUCAACAGGUUCU-3'
P3- DNA sequence dna complementary strand (P3-DNA)：5'-FAM-AGAACCTGTTGATTCAACAGCACCA-3'
(2) the copper metal coordination polymer aqueous solution is prepared
Copper metal coordination polymer is dissolved in distilled water and is configured to solution that concentration is 250mM and with sterilizing filter pair Solution carries out degerming, and sealing preserve is stand-by as stock solution.
(3) metal coordinating polymer is to fluorescence labeling P3The fluorescent quenching of-DNA
With 100 μM of Tris-HCl cushioning liquid by 100 μM of P3- DNA solution is diluted to 50nM, used as liquid to be titrated；Take Metal coordinating polymer stock solution is diluted to 500 μM of (P containing 50nM with 100 μM of Tris-HCl cushioning liquid3- DNA) as drop Determine liquid.Take P3Titrating solution in fluorescence cuvette is pressed volume gradient and is added in cuvette, record body by-DNA liquid 2mL to be titrated It is the change (λ of fluorescence intensityex=480nm) until fluorescent quenching reaches saturation, calculate fluorescent quenching efficiency QE%.The copper metal Coordination polymer is to fluorescently-labeled P3The fluorescent quenching rate of-DNA is 91.4%.
(4) compound system is to T to be checked3The recognition detection of-RNA sequence
On above-mentioned experiment basis, by dilution after T to be checked3- RNA solution (10 μM) is added to fluorescent quenching and reaches saturation Compound system in, make T2- RNA reaches Cmin 25nM in system, and monitoring fluorescence intensity over time, determines body It is that fluorescence recovery reaches the time of maximum as detection T3The reaction time R of-RNAT.T is detected with compound system3- RNA's is anti- R between seasonableTFor standard, record compound system fluorescence intensity is with T3- RNA concentration increases and the extent of recovery, until fluorescence recovery reaches To saturation, fluorescence anabiosis rate R is calculatedEAnd test limit.Copper metal coordination polymer and fluorescently-labeled P3- DNA forms compound system It is 20min, fluorescence anabiosis rate R to 2 type nucleic acid sequence of the dengue fever detection reaction timeEFor 3.6, detection is limited to 51pM.Should As a result show both sexes carboxylic acid of the present invention three-dimensional copper metal coordination polymer 2 type nucleic acid sequence of dengue fever is had compared with Good detection effect.
SEQUENCE LISTING
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<120>A kind of both sexes carboxylic acid 3-dimensional metal coordination polymer and its preparation method and application
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Claims (8)

1. soluble amphoteric carboxylic acid 3-dimensional metal coordination polymer, it is characterised in that the master of three-dimensional copper metal coordination polymer matches somebody with somebody Body is N- (3,5- dicarboxyl benzyls)-(3- carboxyls)-pyridinium bromide (H3DcbcpBr), assistant ligand is 1,2-, bis- (4- pyridines Base) ethene (bpe), central atom is copper.
2. soluble amphoteric carboxylic acid 3-dimensional metal coordination polymer according to claim 1, by its constitutional repeating unit [Cu (Dcbcp) monoclinic system for (bpe)] constituting, with three-dimensional structure.
3. the soluble amphoteric carboxylic acid 3-dimensional metal coordination polymer according to any one of claim 1-2, each two Cu are former Son is coordinated to form [Cu with Dcbcp2(Dcbcp)4] secondary structure, [a Cu2(Dcbcp)4] secondary structure is identical with surrounding six Structure connection, formed on bc axial planes two dimension planar structure.
4. the soluble amphoteric carboxylic acid 3-dimensional metal coordination polymer according to any one of claim 1-3, it is characterised in that Comprise the following steps：
(1) N- (3,5- dicarboxyl benzyl)-(3- carboxyls)-pyridinium bromide is scattered in distilled water, adjusts pH to 7.0, led Ligand solution；
(2) will be dissolved in the water with the copper sulphate of N- (3,5- dicarboxyl benzyl)-(3- carboxyls)-pyridinium bromide equimolar amounts, obtain Arrive copper salt solution；
(3) will be with 1,2- bis- (4- pyridine radicals) ethene of N- (3,5- dicarboxyl benzyls)-(3- carboxyls)-pyridinium bromide equimolar amounts It is dissolved in DMF, obtains assistant ligand solution；
(4) copper salt solution that step (2) is obtained is added to step (1) to obtain in main ligand solution, mistake after reaction is stirred at room temperature Filter, the assistant ligand solution for adding step (3) to obtain in filtrate, filters after stirring, and filtrate temperature programming reacts 72h to 100 DEG C, Programmed cooling separates out green lump shaped crystalline to 30 DEG C again afterwards, collects crystal, is vacuum dried after washed with ether, obtains described two Property copper carboxylate metal coordinating polymer [Cu (Dcbcp) is (bpe)].
5. the soluble amphoteric carboxylic acid 3-dimensional metal coordination polymer according to any one of claim 1-3 is used as fluorescent dye Quencher, it is preferable that fluorescent dye be selected from FAM, ROX, CY5.
6. a kind of detection nucleic acid method, it is characterised in that by with target nucleic acid fluorescent dye groups and to be measured can specificity In conjunction with probe molecule mix with the soluble amphoteric carboxylic acid 3-dimensional metal coordination polymer described in any one of claim 1-3, Testing sample is subsequently adding, and detects fluorescence.
7. a kind of detection nucleic acid reagent, which includes probe molecule that is mixing or being discretely located and metal coordination polymerization Thing, wherein probe molecule are the probe molecule that can be specifically bound with target nucleic acid fluorescent dye groups and to be measured, and metal is matched somebody with somebody Position polymer is the soluble amphoteric carboxylic acid 3-dimensional metal coordination polymer described in any one of claim 1-3.
8. application of the reagent of detection nucleic acid according to claim 7 in detection detection virus agent box is prepared, preferably Ground, the virus are selected from dengue fever virus, AIDS virus or Sudan virus.
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CN109929118A (en) * 2019-03-29 2019-06-25 上海大学 A kind of Cu(I) coordination polymer and the preparation method and application thereof
CN112961294A (en) * 2021-02-05 2021-06-15 河南大学 Water-soluble MOF composite polymer material and preparation method and application thereof

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CN105906817A (en) * 2016-06-08 2016-08-31 南方医科大学 Amphoteric carboxylic acid hexanuclear copper metal coordination polymer and preparation method thereof
CN106084245A (en) * 2016-06-08 2016-11-09 南方医科大学 A kind of both sexes carboxylic acid four core copper metal coordinating polymer and preparation method thereof

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CN101932707A (en) * 2008-01-31 2010-12-29 库瑞瓦格有限责任公司 Nucleic acids of formula (i) (nuglxmgnnv)a and derivatives thereof as an immunostimulating agent/adjuvant
CN105566653A (en) * 2015-12-16 2016-05-11 南方医科大学 Amphiprotic lanthanum carboxylate metal organic framework and synthesis method thereof, and application of amphiprotic lanthanum carboxylate metal organic framework
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CN106084245A (en) * 2016-06-08 2016-11-09 南方医科大学 A kind of both sexes carboxylic acid four core copper metal coordinating polymer and preparation method thereof

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* Cited by examiner, † Cited by third party
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CN109929118A (en) * 2019-03-29 2019-06-25 上海大学 A kind of Cu(I) coordination polymer and the preparation method and application thereof
CN109929118B (en) * 2019-03-29 2022-04-19 上海大学 Cu (I) coordination polymer and preparation method and application thereof
CN112961294A (en) * 2021-02-05 2021-06-15 河南大学 Water-soluble MOF composite polymer material and preparation method and application thereof

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