CN118222288A - PtAg alloy nanocluster and preparation method and application thereof - Google Patents
PtAg alloy nanocluster and preparation method and application thereof Download PDFInfo
- Publication number
- CN118222288A CN118222288A CN202410322295.8A CN202410322295A CN118222288A CN 118222288 A CN118222288 A CN 118222288A CN 202410322295 A CN202410322295 A CN 202410322295A CN 118222288 A CN118222288 A CN 118222288A
- Authority
- CN
- China
- Prior art keywords
- dppb
- solution
- hcy
- dichloromethane
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229910045601 alloy Inorganic materials 0.000 title claims abstract description 21
- 239000000956 alloy Substances 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- BCJVBDBJSMFBRW-UHFFFAOYSA-N 4-diphenylphosphanylbutyl(diphenyl)phosphane Chemical compound C=1C=CC=CC=1P(C=1C=CC=CC=1)CCCCP(C=1C=CC=CC=1)C1=CC=CC=C1 BCJVBDBJSMFBRW-UHFFFAOYSA-N 0.000 claims abstract description 74
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Substances [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 97
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 81
- 239000000243 solution Substances 0.000 claims description 51
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 claims description 50
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 45
- 239000011259 mixed solution Substances 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 9
- 239000012043 crude product Substances 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- -1 2,3,4,5, 6-pentafluorophenyl thiophenol Chemical compound 0.000 claims description 8
- 238000005580 one pot reaction Methods 0.000 claims description 8
- 238000006722 reduction reaction Methods 0.000 claims description 8
- QBVXKDJEZKEASM-UHFFFAOYSA-M tetraoctylammonium bromide Chemical compound [Br-].CCCCCCCC[N+](CCCCCCCC)(CCCCCCCC)CCCCCCCC QBVXKDJEZKEASM-UHFFFAOYSA-M 0.000 claims description 8
- 230000009194 climbing Effects 0.000 claims description 7
- 229910052697 platinum Inorganic materials 0.000 claims description 7
- 229910052709 silver Inorganic materials 0.000 claims description 7
- 239000004332 silver Substances 0.000 claims description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- GKFJEDWZQZKYHV-UHFFFAOYSA-N borane;2-methylpropan-2-amine Chemical compound B.CC(C)(C)N GKFJEDWZQZKYHV-UHFFFAOYSA-N 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 239000000741 silica gel Substances 0.000 claims description 6
- 229910002027 silica gel Inorganic materials 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- GLJFYGFBITUZOE-UHFFFAOYSA-N 4-phenylbutylbenzene Chemical compound C=1C=CC=CC=1CCCCC1=CC=CC=C1 GLJFYGFBITUZOE-UHFFFAOYSA-N 0.000 claims description 4
- 238000002425 crystallisation Methods 0.000 claims description 4
- 230000008025 crystallization Effects 0.000 claims description 4
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical group [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 4
- GYCDHQVDDJPULY-UHFFFAOYSA-N (2,3,4,5-tetrafluoro-6-phenylphenyl) thiohypofluorite Chemical compound FSC1=C(C(=C(C(=C1F)F)F)F)C1=CC=CC=C1 GYCDHQVDDJPULY-UHFFFAOYSA-N 0.000 claims description 3
- JYHRLWMNMMXIHF-UHFFFAOYSA-N (tert-butylamino)boron Chemical compound [B]NC(C)(C)C JYHRLWMNMMXIHF-UHFFFAOYSA-N 0.000 claims description 2
- QKLWAMMQKBOTCD-UHFFFAOYSA-N butane;diphenylphosphane Chemical group CCCC.C=1C=CC=CC=1PC1=CC=CC=C1 QKLWAMMQKBOTCD-UHFFFAOYSA-N 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 2
- 239000013049 sediment Substances 0.000 claims 3
- 238000005119 centrifugation Methods 0.000 claims 1
- 238000004506 ultrasonic cleaning Methods 0.000 claims 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 20
- 239000001301 oxygen Substances 0.000 abstract description 20
- 229910052760 oxygen Inorganic materials 0.000 abstract description 20
- 229910052751 metal Inorganic materials 0.000 abstract description 13
- 239000002184 metal Substances 0.000 abstract description 13
- 239000003446 ligand Substances 0.000 abstract description 9
- 230000004044 response Effects 0.000 abstract description 7
- 230000005284 excitation Effects 0.000 abstract description 6
- 239000002131 composite material Substances 0.000 abstract description 3
- 239000002086 nanomaterial Substances 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 13
- 239000002244 precipitate Substances 0.000 description 13
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 12
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 12
- 239000013078 crystal Substances 0.000 description 11
- RGSFGYAAUTVSQA-UHFFFAOYSA-N Cyclopentane Chemical compound C1CCCC1 RGSFGYAAUTVSQA-UHFFFAOYSA-N 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 238000010521 absorption reaction Methods 0.000 description 6
- 238000004140 cleaning Methods 0.000 description 6
- 235000018417 cysteine Nutrition 0.000 description 6
- 229960003180 glutathione Drugs 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 6
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 5
- 229910003460 diamond Inorganic materials 0.000 description 5
- 239000010432 diamond Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 5
- DMEGYFMYUHOHGS-UHFFFAOYSA-N heptamethylene Natural products C1CCCCCC1 DMEGYFMYUHOHGS-UHFFFAOYSA-N 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108010024636 Glutathione Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000001308 synthesis method Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 description 3
- YCKRFDGAMUMZLT-UHFFFAOYSA-N Fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000000695 excitation spectrum Methods 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 229910052731 fluorine Inorganic materials 0.000 description 2
- 239000011737 fluorine Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 125000004430 oxygen atom Chemical group O* 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000006862 quantum yield reaction Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- HXJUTPCZVOIRIF-UHFFFAOYSA-N sulfolane Chemical compound O=S1(=O)CCCC1 HXJUTPCZVOIRIF-UHFFFAOYSA-N 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 2
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 2
- KUAPPJSILOMQPC-UHFFFAOYSA-N 2-chloro-4-fluorobenzenethiol Chemical compound FC1=CC=C(S)C(Cl)=C1 KUAPPJSILOMQPC-UHFFFAOYSA-N 0.000 description 1
- 229910001316 Ag alloy Inorganic materials 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 241000790917 Dioxys <bee> Species 0.000 description 1
- 229910001260 Pt alloy Inorganic materials 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- MJYGPNBYCMSMFL-UHFFFAOYSA-N [O].C1CCOC1 Chemical group [O].C1CCOC1 MJYGPNBYCMSMFL-UHFFFAOYSA-N 0.000 description 1
- HRQWCSQSHDRRCQ-UHFFFAOYSA-N [O].S1(=O)(=O)CCCC1 Chemical group [O].S1(=O)(=O)CCCC1 HRQWCSQSHDRRCQ-UHFFFAOYSA-N 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- VURFVHCLMJOLKN-UHFFFAOYSA-N diphosphane Chemical compound PP VURFVHCLMJOLKN-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 238000005401 electroluminescence Methods 0.000 description 1
- 238000000119 electrospray ionisation mass spectrum Methods 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000005389 magnetism Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005424 photoluminescence Methods 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000022558 protein metabolic process Effects 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a PtAg alloy nanocluster, and a preparation method and application thereof, belonging to the technical field of nano materials. The Pt 1Ag12 metal core is covered by two Ag (C 6F5S)3 staples to form a composite structure, and then is surrounded by three DPPB ligands to form an integral structure, the Pt 1Ag14(DPPB)3(C6F5S)6 nanocluster prepared by the invention has good fluorescence response to Hcy, pt 1Ag14(DPPB)3(C6F5S)6 has better Hcy recognition capability under long wavelength excitation in the presence of oxygen, and has higher contrast ratio compared with single photon fluorescence.
Description
Technical Field
The invention relates to the technical field of nano materials, in particular to a PtAg alloy nanocluster, and a preparation method and application thereof.
Background
Metal Nanoclusters (MNCs) are ultra-small particles with special photophysical and chemical properties, exhibiting rich material properties such as fluorescence, chirality, magnetism, catalysis, electrochemistry, electroluminescence, electrochemiluminescence, etc. In recent years, many top chemists in the chemical field have conducted extensive and intensive studies on metal nanoclusters having a photoluminescent effect. Metal nanoclusters with strong fluorescence begin to be used as probe molecules for the detection of bio-specific molecules.
Homocysteine HCy (homocysteine) is an amino acid, and the content in human bodies is related to the occurrence of cardiovascular diseases, apoplexy, arteriosclerosis and other diseases. Therefore, detection of homocysteine is of great importance in clinical diagnosis. Cysteine (Cys) is also a critical amino acid that plays an important role in redox homeostasis, protein function and metabolism. In laboratory detection, due to the lack of a more reliable, faster and more convenient test detection method, how to distinguish between general cysteines (Cys), homocysteine (Hcy), glutathione (GSH) and the like in the same detection time is still a significant and difficult challenge in a complex redox environment. In addition, it is also important to visualize the results of dynamic biological events occurring in vivo and in vitro during the real-time monitoring of probe detection, thereby accurately reflecting the temporal and spatial processes. Therefore, the invention provides a PtAg alloy nanocluster, and a preparation method and application thereof.
Disclosure of Invention
The invention provides a Pt/Ag alloy nanocluster, a preparation method and application thereof, which effectively solve the technical problem that common cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and the like cannot be effectively distinguished in the same detection time in a complex redox environment, and simultaneously provide a strong fluorescence Pt 1Ag14(DPPB)3(C6F5S)6 cluster capable of enhancing the detection behavior of Hcy in an oxygen environment.
The first object of the present invention is to provide a PtAg alloy nanocluster having a molecular formula of Pt 1Ag14(DPPB)3(C6F5S)6, wherein DPPB is diphenylphosphine butane and C 6F5 S is pentafluorophenyl thiophenol.
The second object of the present invention is to provide a preparation method of PtAg alloy nanoclusters, including the following steps:
Dissolving tetra-n-octyl ammonium bromide in a mixed solution of dichloromethane and methanol, adding a silver source aqueous solution, stirring, adding a platinum source aqueous solution, stirring, sequentially adding 2,3,4,5, 6-pentafluorophenyl thiophenol and 1, 4-diphenylbutane for initial reaction to obtain a mixed solution, adding a methanol solution of tert-butylamine borane into the mixed solution, carrying out one-pot reduction reaction to obtain a crude product, and crystallizing to obtain PtAg alloy nanoclusters.
As a preferred embodiment, the tetra-n-octylammonium bromide, silver source, platinum source, 2,3,4,5, 6-pentafluorophenylthiophenol, 1, 4-bis-diphenylbutane and t-butylamino borane are used in a ratio of 2mg to 4.59. Mu. Mol 0.49. Mu. Mol 1uL to 1mg to 4mg.
As a preferred embodiment, the silver source is silver nitrate.
As a preferred embodiment, the platinum source is chloroplatinic acid.
As a preferred embodiment, the one-pot reduction reaction takes 10 to 12 hours.
As a preferred embodiment, the initial reaction time is 15 to 30 minutes.
As a preferred embodiment, prior to crystallization, the crude product is dissolved with dichloromethane, added with ethanol, mixed and shaken uniformly, ultrasonically washed, centrifuged, the precipitate is collected, dissolved with dichloromethane, and applied to TLC silica gel plates for purification by climbing plates.
The third object of the invention is to provide an application of the PtAg alloy nanocluster in detection of homocysteine.
Compared with the prior art, the invention has the beneficial effects that:
The invention provides a PtAg alloy nanocluster and a preparation method and application thereof, wherein tetra-n-octyl ammonium bromide is dissolved in a mixed solution of dichloromethane and methanol, a silver source aqueous solution is added, stirring is carried out, a platinum source aqueous solution is added, stirring is carried out, 2,3,4,5, 6-pentafluorophenyl thiophenol and 1, 4-diphenyl butane are sequentially added for initial reaction, a mixed solution is obtained, a methanol solution of tert-butylamine borane is added into the mixed solution, one-pot reduction reaction is carried out, a crude product is obtained, and crystallization is carried out, thus obtaining the PtAg alloy nanocluster.
The PtAg alloy nanocluster prepared by the method, namely Pt 1Ag14(DPPB)3(C6F5S)6, has a platinum atom and 12 silver atoms to jointly form an icosahedron metal core. The Pt 1Ag12 metal core is covered by two Ag (C 6F5S)3 staples to form a composite structure, and then is surrounded by three DPPB ligands to form an integral structure, the Pt 1Ag14(DPPB)3(C6F5S)6 nanocluster prepared by the invention has good fluorescence response to Hcy, pt 1Ag14(DPPB)3(C6F5S)6 has better Hcy recognition capability under long wavelength excitation in the presence of oxygen, and has higher contrast ratio compared with single photon fluorescence.
Drawings
FIG. 1 is a diagram of the summarized structure of Pt1Ag14(DPPB)3(C6F5S)6、Pt1Ag14(DPPB)3(C6F5S)6- sulfolane molecules and Pt1Ag14(C6HF5S)6(DPPB)3-(C4H9O2NS)-(CH2Cl2) prepared according to the present invention;
FIG. 2 is a structural exploded view of Pt 1Ag14(DPPB)3(C6F5S)6 prepared in accordance with the present invention;
FIG. 3 is an ultraviolet-visible spectrum (dissolved in CH 2Cl2) of orange diamond-shaped bulk Pt 1Ag14(DPPB)3(C6F5S)6 single crystals prepared in example 1 of the present invention;
FIG. 4 is a graph of ESI-MS data for Pt 1Ag14(DPPB)3(C6F5S)6 prepared in example 1 of the present invention;
FIG. 5 is a graph showing the excitation spectrum and the emission spectrum of Pt 1Ag14(DPPB)3(C6F5S)6 prepared in example 1 of the present invention;
FIG. 6 is a graph showing the solid fluorescence lifetime of Pt 1Ag14(DPPB)3(C6F5S)6 prepared in example 1 of the present invention;
FIG. 7 is a graph of fluorescence lifetime of Pt 1Ag14(DPPB)3(C6F5S)6 (in CH 2Cl2 solution) prepared in example 1 of the present invention;
FIG. 8 is a graph showing the fluorescence response of Pt 1Ag14(DPPB)3(C6F5S)6 to Hcy prepared in example 1 of the present invention;
FIG. 9 is a graph showing the change in fluorescence intensity of Pt 1Ag14(DPPB)3(C6F5S)6 prepared in example 1 according to the present invention after oxygen is introduced;
FIG. 10 is a graph showing the fluorescence response of Pt 1Ag14(DPPB)3(C6F5S)6 prepared in example 1 of the present invention to Hcy under an oxygen atmosphere;
FIG. 11 is a graph showing specific recognition of Hcy and Cys by Pt 1Ag14(DPPB)3(C6F5S)6 prepared in example 1 of the present invention;
FIG. 12 is a graph showing the change in three-photon fluorescence intensity of Pt 1Ag14(DPPB)3(C6F5S)6 prepared in example 1 according to the present invention as the oxygen content increases;
FIG. 13 is a graph showing the variation of the fluorescence intensity of Pt 1Ag14(DPPB)3(C6F5S)6 prepared in example 1 according to the present invention as the Hcy concentration increases;
FIG. 14 is a three-photon absorption cross-sectional view of Pt 1Ag14(DPPB)3(C6F5S)6 prepared in example 1 of the present invention;
FIG. 15 is a graph showing the effect of an increase in Hcy under an oxygen atmosphere on the three-photon fluorescence intensity of Pt 1Ag14(DPPB)3(C6F5S)6 prepared in example 1 of the present invention;
FIG. 16 is a graph showing the effect of an increase in Hcy under an oxygen atmosphere on the three-photon absorption cross section of Pt 1Ag14(DPPB)3(C6F5S)6 prepared in example 1 of the present invention.
Detailed Description
In order that those skilled in the art will better understand the technical solution of the present invention, the present invention will be further described with reference to the specific examples and the accompanying drawings, but the examples are not intended to be limiting. The following test methods and detection methods, if not specified, are conventional methods; the reagents and starting materials, unless otherwise specified, are commercially available.
Aiming at the technical problems that common cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and the like cannot be distinguished within the same detection time in the prior art, and the results of dynamic biological events which occur in vivo and in vitro cannot be visualized in the process of real-time monitoring by a probe detection, so that time and space processes are accurately reflected, the invention provides a PtAg alloy nanocluster and a preparation method and application thereof.
The Pt 1Ag14(DPPB)3(C6F5S)6 nanocluster prepared by the method has good fluorescence response to Hcy, pt 1Ag14(DPPB)3(C6F5S)6 has better Hcy recognition capability under long-wavelength excitation in the presence of oxygen, and compared with single photon fluorescence, the Pt 1Ag14(DPPB)3(C6F5S)6 nanocluster has higher contrast.
Effects will be described below with reference to specific examples.
Example 1
A preparation method of PtAg alloy nanoclusters comprises the following steps:
The strong fluorescent Pt 1Ag14(DPPB)3(C6F5S)6 nanocluster is prepared by adopting a one-pot reduction synthesis method:
S1, 100mg of tetra-n-octyl ammonium bromide is dissolved in a mixed solution of 15mL of dichloromethane and 5mL of methanol, and the mixed solution is uniformly vibrated to obtain a uniform solution; 50mgAgNO 3 of the solution is dissolved in 2mL of H 2 O, added into the uniform solution and stirred for 5min, and the solution is changed into a pale yellow clear solution from colorless and transparent;
S2, adding 10mg of chloroplatinic acid aqueous solution into the clear light yellow solution, stirring for 10min, gradually changing the solution into a turbidous solution, adding 50 mu L of 2,3,4,5, 6-pentafluorophenyl thiophenol into the turbidimetric solution after 20min, slowly changing the solution into dark green, adding 50mg of 1, 4-bisdiphenylbutane after 15min, gradually changing the solution into a light green turbid solution from turbidimetric solution, performing initial reaction for 30min to obtain a mixed solution, dissolving 200mg of tert-butylamine borane (C 4H11 BN) into 2mL of CH 3 OH, pouring into the mixed solution, continuously reacting for 12h, slowly changing the solution system from black to transparent orange clear liquid and slowly generating orange fluorescent light, centrifuging for 3min at a high rotating speed of 11000rpm, and removing black impurity precipitate to obtain orange clear liquid;
S3, spin-drying orange supernatant to obtain orange crude product, dissolving with 2mL of dichloromethane, adding 2mL of ethanol, mixing, shaking uniformly, ultrasonically cleaning for 3min until orange precipitate is generated, centrifuging, taking precipitate, cleaning the precipitate, collecting orange powder, dissolving orange powder with dichloromethane, coating and loading on a TLC silica gel plate for climbing plate purification (toluene/cyclopentane=1:1), and crystallizing and diffusing at room temperature with a dichloromethane/methanol mixed system to obtain orange transparent diamond monocrystal, namely Pt 1Ag14(DPPB)3(C6F5S)6 nanoclusters.
Example 2
A preparation method of PtAg alloy nanoclusters comprises the following steps:
The strong fluorescent Pt 1Ag14(DPPB)3(C6F5S)6 nanocluster is prepared by adopting a one-pot reduction synthesis method:
S1, 100mg of tetra-n-octyl ammonium bromide is dissolved in a mixed solution of 15mL of dichloromethane and 5mL of methanol, and the mixed solution is uniformly vibrated to obtain a uniform solution; 50mgAgNO 3 of the solution is dissolved in 2mL of H 2 O, added into the uniform solution and stirred for 5min, and the solution is changed into a pale yellow clear solution from colorless and transparent;
S2, adding 10mg of chloroplatinic acid aqueous solution into the clear light yellow solution, stirring for 10min, gradually changing the solution into a turbidous solution, adding 45 mu L of 2,3,4,5, 6-pentafluorophenyl thiophenol into the turbidimetric solution after 20min, slowly changing the solution into dark green, adding 45mg of 1, 4-bisdiphenylbutane after 15min, gradually changing the solution into a light green turbid solution from turbidimetric solution, performing initial reaction for 15min to obtain a mixed solution, dissolving 180mg of tert-butylamine borane (C 4H11 BN) into 2mL of CH 3 OH, pouring into the mixed solution, continuously reacting for 10h, slowly changing the solution system from black to transparent orange clear liquid and slowly generating orange fluorescent light, centrifuging for 3min at a high rotating speed of 11000rpm, and removing black impurity precipitate to obtain orange clear liquid;
S3, spin-drying orange supernatant to obtain orange crude product, dissolving with 2mL of dichloromethane, adding 2mL of ethanol, mixing, shaking uniformly, ultrasonically cleaning for 3min until orange precipitate is generated, centrifuging, taking precipitate, cleaning the precipitate, collecting orange powder, dissolving orange powder with dichloromethane, coating and loading on a TLC silica gel plate for climbing plate purification (toluene/cyclopentane=1:1), and crystallizing and diffusing at room temperature with a dichloromethane/methanol mixed system to obtain orange transparent diamond monocrystal, namely Pt 1Ag14(DPPB)3(C6F5S)6 nanoclusters.
Example 3
A preparation method of PtAg alloy nanoclusters comprises the following steps:
The strong fluorescent Pt 1Ag14(DPPB)3(C6F5S)6 nanocluster is prepared by adopting a one-pot reduction synthesis method:
S1, 100mg of tetra-n-octyl ammonium bromide is dissolved in a mixed solution of 15mL of dichloromethane and 5mL of methanol, and the mixed solution is uniformly vibrated to obtain a uniform solution; 50mgAgNO 3 of the solution is dissolved in 2mL of H 2 O, added into the uniform solution and stirred for 5min, and the solution is changed into a pale yellow clear solution from colorless and transparent;
s2, adding 10mg of chloroplatinic acid aqueous solution into the clear light yellow solution, stirring for 10min, gradually changing the solution into a turbidous solution, adding 55 mu L of 2,3,4,5, 6-pentafluorophenyl thiophenol into the turbidimetric solution after 20min, slowly changing the solution into dark green, adding 55mg of 1, 4-bisdiphenylbutane after 15min, gradually changing the solution into a light green turbid solution from turbidimetric solution, performing initial reaction for 20min to obtain a mixed solution, dissolving 220mg of tert-butylamine borane (C 4H11 BN) into 2mL of CH 3 OH, pouring into the mixed solution, continuously reacting for 11h, slowly changing the solution system from black to transparent orange clear liquid and slowly generating orange fluorescent light, centrifuging for 3min at a high rotating speed of 11000rpm, and removing black impurity precipitate to obtain orange clear liquid;
S3, spin-drying orange supernatant to obtain orange crude product, dissolving with 2mL of dichloromethane, adding 2mL of ethanol, mixing, shaking uniformly, ultrasonically cleaning for 3min until orange precipitate is generated, centrifuging, taking precipitate, cleaning the precipitate, collecting orange powder, dissolving orange powder with dichloromethane, coating and loading on a TLC silica gel plate for climbing plate purification (toluene/cyclopentane=1:1), and crystallizing and diffusing at room temperature with a dichloromethane/methanol mixed system to obtain orange transparent diamond monocrystal, namely Pt 1Ag14(DPPB)3(C6F5S)6 nanoclusters.
The performance of the Pt 1Ag14(DPPB)3(C6F5S)6 nano-cluster prepared by the method is tested, and the specific process and the result are as follows:
To verify that the fluorine-containing ligand of Pt 1Ag14(DPPB)3(C6F5S)6 prepared according to the present invention has the ability to adsorb oxygen-containing molecules, pt 1Ag14(C6F5S)6(DPPB)3@(C4H8O2 S) nanoclusters and Pt1Ag14(C6HF5S)6(DPPB)3-(C4H9O2NS)-(CH2Cl2) nanoclusters were also synthesized according to the present invention, specifically as follows:
1. Synthesis of the diamond Pt1Ag14(DPPB)3(C6F5S)6@(C4H8O2S) nm cluster containing dioxy orange red: the purified Pt 1Ag14(C6HF5S)6(DPPB)3 was dissolved in 4mL of dichloromethane and concentrated to a long single crystal concentration and 1mL of sulfolane solvent was added. Shaking the mixture solution, removing particle impurities by using a filter head, and crystallizing the pure orange Pt1Ag14(C6F5S)6(DPPB)3@(C4H8O2S) concentrated stock solution by using dichloromethane/methanol to obtain the orange regular diamond monocrystal.
2.Pt1Ag14(C6HF5S)6(DPPB)3-(C4H9O2NS)-(CH2Cl2) Synthesis of nanoclusters
Pt1Ag14(C6HF5S)6(DPPB)3@(C4H9O2NS) Nanocluster synthesis containing homocysteine molecule (Hcy): the crude product concentrated solution containing Pt 1Ag14(C6HF5S)6(DPPB)3 nanoclusters was carried on TLC silica gel plates for plate climbing purification (toluene/cyclopentane=1:1). And performing diffusion crystallization on the orange-red pure Pt 1Ag14(C6HF5S)6(DPPB)3 after climbing the plate by using dichloromethane/methanol at the room temperature of 25 ℃ to obtain a blocky orange-red monocrystal. After two to three weeks, the red single crystals in the form of blocks were sucked out and immersed in the now prepared mixed solution (the mixed solution was first composed of 2ml of ultrapure water and 2ml of acetonitrile in which 20mg of homocysteine Hcy was dissolved).
The presence of the carboxyl group, an oxygen-containing functional group, in the formula of homocysteine (Hcy), and Pt1Ag14(C6F5S)6(DPPB)3@(C4H8O2S) prepared as described above, indicates that the sulfolane oxygen atom has an oxyfluoride bond with F in the pentafluorophenylthiol ligand. By the same reason ,Pt1Ag14(C6HF5S)6(DPPB)3-(C4H9O2NS)-(CH2Cl2), the interaction force between the tetrahydrofuran oxygen atom and fluorine in the pentafluorophenyl thiophenol ligand is verified through the single crystal structure, and the preparation of the two single crystals lays a theoretical foundation for subsequent fluorescence detection. The combination of PtAg alloy nanoclusters and homocysteine prepared by the method is oxyfluoride acting force generated near 5F thiophenol ligand, thereby verifying that sulfolane containing two oxygen atoms or one oxygen-containing molecule tetrahydrofuran grows from a single crystal near the pentafluoropthiophenol ligand and Hcy containing carboxyl of the oxygen atom at last; the single crystal data lay a theoretical basis for the subsequent fluorescence detection.
The overall structures of the Pt 1Ag14(C6F5S)6(DPPB)3 nanoclusters prepared in example 1 of the present invention and the two nanoclusters described above are shown in fig. 1 and 2.
The overall structure of Pt 1Ag14(DPPB)3(C6F5S)6 is shown in fig. 1 and 2: together, one platinum atom and 12 silver atoms make up an icosahedral metal core. The Pt 1Ag12 metal core was then first covered with two Ag (C 6F5S)3 staples to form a composite structure and then surrounded by three DPPB ligands to form a monolithic structure unlike the Pt 1Ag14(SR)6(PPh3)8 (H-SR: 2-chloro-4-fluorobenzene thiol) reported in the previous article, the average Ag-Ag bond distance between the Pt 1Ag12 metal core in Pt 1Ag14(SR)6(PPh3)8 and the Ag (C 6H3FClS)3(PPh3) staple motif was about as long as the silver-silver bond between the Pt 1Ag12 core structure and the Ag (C 6F5S)3 staple motif was changed due to the absence of the biphosphine ligand on the Ag (C 6F5S)3 staple)While the Ag-Ag bond distance between the Pt 1Ag14(DPPB)3(C6F5S)6 metal core Pt 1Ag12 and the short staple motif Ag (C 6F5S)3) is aboutThe composition structure promotes Ag-Ag metallophilic interaction in the metal nanoclusters.
Ultraviolet-visible Spectrum of Pt 1Ag14(DPPB)3(C6F5S)6
The ultraviolet spectrum of the orange diamond-shaped bulk Pt 1Ag14(DPPB)3(C6F5S)6 crystal prepared in example 1 dissolved in 2mL of dichloromethane is shown in FIG. 3, the nanoclusters have obvious absorption peaks at 400nm and 520, and a weaker absorption peak is further shown at 460 nm.
3. Electrospray ionization mass spectrometry (ESI) of Pt 1Ag14(DPPB)3(C6F5S)6
The Pt 1Ag14(DPPB)3(C6F5S)6 crystals were dissolved in a mixed solution of dichloromethane and methanol for electrospray ionization mass spectrometry. In the ESI-MS spectrum we found a signal peak at m/z= 4201.9730Da with a 1Da interval, which is assigned to signal peak [Pt1Ag14(C28H28P2)3(C6F5S)6Na]+, as shown in fig. 4. This experimental result confirms that the Pt 1Ag14(DPPB)3(C6F5S)6 metal nanocluster is neutral. And combining the crystal data with ESI-MS results, and verifying the accuracy of the structure.
4. Investigation of fluorescent Properties of Pt 1Ag14(DPPB)3(C6F5S)6
Since Pt 1Ag14(DPPB)3(C6F5S)6 crystals have strong red fluorescence under uv irradiation, we studied the photoluminescence properties of Pt 1Ag14(DPPB)3(C6F5S)6, and first Pt 1Ag14(DPPB)3(C6F5S)6 exhibited red fluorescence under uv irradiation. Pt 1Ag14(DPPB)3(C6F5S)6 was dissolved in dichloromethane solvent and excited at 400nm, the emission peak of Pt 1Ag14(DPPB)3(C6F5S)6 was at 620nm, as shown in fig. 5. By excitation at 620nm, an excitation spectrum of Pt 1Ag14(DPPB)3(C6F5S)6 was obtained, with signal peaks at 523nm, 402nm and 375nm, similar to the UV-visible absorption spectrum of the metal nanocluster liquid. The fluorescence lifetime of PtAg 14(DPPB)3(C6F5S)6 was 2.75us in CH 2Cl2 solution and 3.44us in solid, both in liquid and solid state, had long lifetimes, with Quantum Yields (QY) of about 47.32% and 58.75%, respectively, as shown in fig. 6 and 7. The remarkable fluorescent properties give clusters the potential to detect small molecules efficiently.
5. Application of Pt 1Ag14(DPPB)3(C6F5S)6 in detection of homocysteine
Investigation of the optical properties of Pt 1Ag14(DPPB)3(C6F5S)6 for detecting homocysteine (Hcy) by oxygen gas introduction fig. 8 is a graph showing the fluorescence response of Pt 1Ag14(DPPB)3(C6F5S)6 prepared in example 1 of the present invention to Hcy, with increasing Hcy concentration (from 0 μm to 400 μm), the fluorescence intensity increased 1.3 times compared to the blank, indicating that Pt 1Ag14(DPPB)3(C6F5S)6 nanoclusters have good fluorescence response to Hcy.
FIG. 9 is a graph showing the change in fluorescence intensity of Pt 1Ag14(DPPB)3(C6F5S)6 prepared in example 1 according to the present invention after oxygen is introduced; after oxygen is introduced, the fluorescence intensity of the cluster is obviously reduced, and the oxygen has quenching effect on the fluorescence intensity of the cluster. After oxygen is introduced, hcy (from 0 mu M to 400 mu M) is added dropwise, and the fluorescence intensity is gradually increased, as shown in fig. 10, the fluorescence intensity is increased by 2 times, and after oxygen is introduced, the recognition effect of 5F-Pt 1Ag14 on Hcy is remarkably improved compared with that of the case of oxygen. FIG. 11 is a specific recognition Hcy experiment, cys (cysteine) and Hcy (homocysteine) chemical structures and properties are very similar, and thus specific detection of these two substances is very challenging. The DCM mother liquor of 5F-Pt 1Ag14 (c= -3 mol/L) was taken, Diluted to 10 -5 mol/L with DCM, 300. Mu.L (c= -5 mol/L) of Cys (cysteine) and Hcy (homocysteine) were added to the 5F-Pt 1Ag14 DCM solution, respectively, Fluorescence testing is then performed. The fluorescence intensity for recognizing Hcy is 2.4 times that of Cys, and 5F-Pt 1Ag14 can distinguish Hcy from Cys. FIG. 12 illustrates that the three-photon fluorescence intensity of 5F-Pt 1Ag14 gradually decreases with increasing O 2 under long wavelength excitation. FIG. 13 illustrates that as the Hcy concentration (from 0. Mu.M to 300. Mu.M) increases, the three-photon fluorescence intensity of 5F-Pt 1Ag14 gradually increases, the fluorescence intensity after recognition is 1.4 times that before recognition, the three-photon absorption cross section of 5F-Pt 1Ag14 after interaction with Hcy is 3.71X 10 -82cm6s2photon-2, Is 1.3 times that before recognition (fig. 14). FIG. 15 shows that after oxygen is introduced, hcy (concentration from 0. Mu.M to 300. Mu.M) is added dropwise, fluorescence intensity is increased by 9.6 times, three-photon absorption cross section can reach 35.5X10 -82cm6s2photon-2 (FIG. 16) which is 9.3 times that of 5F-Pt 1Ag14, and thus it can be seen that 5F-Pt 1Ag14 has better Hcy recognition capability under long wavelength excitation in the presence of oxygen, and has a higher contrast than single photon fluorescence.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (9)
1. The PtAg alloy nanocluster is characterized by having a molecular formula of Pt 1Ag14(DPPB)3(C6F5S)6, wherein DPPB is diphenylphosphine butane, and C 6F5 S is pentafluorophenyl thiophenol.
2. The preparation method of the PtAg alloy nanocluster is characterized by comprising the following steps:
Dissolving tetra-n-octyl ammonium bromide in a mixed solution of dichloromethane and methanol, adding a silver source aqueous solution, stirring, adding a platinum source aqueous solution, stirring, sequentially adding 2,3,4,5, 6-pentafluorophenyl thiophenol and 1, 4-diphenylbutane for initial reaction to obtain a mixed solution, adding a methanol solution of tert-butylamine borane into the mixed solution, carrying out one-pot reduction reaction to obtain a crude product, and crystallizing to obtain PtAg alloy nanoclusters.
3. The preparation method according to claim 2, wherein the amount of tetra-n-octylammonium bromide, silver source, platinum source, 2,3,4,5, 6-pentafluorophenylthiophenol, 1, 4-diphenylbutane and t-butylamine borane is 2 mg/4.59. Mu. Mol/0.49. Mu. Mol/1 uL/1 mg/4 mg.
4. The method of claim 2, wherein the silver source is silver nitrate.
5. The method of claim 2, wherein the platinum source is chloroplatinic acid.
6. The method according to claim 2, wherein the one-pot reduction reaction is carried out for 10 to 12 hours.
7. The method according to claim 2, wherein the initial reaction time is 15 to 30 minutes.
8. The preparation method according to claim 2, wherein before crystallization, the crude product is dissolved by using dichloromethane, ethanol is added to be mixed and vibrated uniformly, ultrasonic cleaning is performed, centrifugation is performed, sediment is collected, the sediment is dissolved by using dichloromethane, and the sediment is smeared and loaded on a TLC silica gel plate for climbing plate purification.
9. Use of a PtAg alloy nanocluster according to claim 1 for detecting homocysteine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410322295.8A CN118222288A (en) | 2024-03-20 | 2024-03-20 | PtAg alloy nanocluster and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410322295.8A CN118222288A (en) | 2024-03-20 | 2024-03-20 | PtAg alloy nanocluster and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118222288A true CN118222288A (en) | 2024-06-21 |
Family
ID=91510544
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410322295.8A Pending CN118222288A (en) | 2024-03-20 | 2024-03-20 | PtAg alloy nanocluster and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118222288A (en) |
-
2024
- 2024-03-20 CN CN202410322295.8A patent/CN118222288A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dong et al. | A highly selective and sensitive salamo-salen-salamo hybrid fluorometic chemosensor for identification of Zn2+ and the continuous recognition of phosphate anions | |
| US8962339B2 (en) | Fluorescent probe compounds, preparation method and application thereof | |
| Tansakul et al. | Distance-dependent fluorescence quenching and binding of CdSe quantum dots by functionalized nitroxide radicals | |
| Pyle et al. | Synthesis and characterization of physical, electronic, and photochemical aspects of 9, 10-phenanthrenequinonediimine complexes of ruthenium (II) and rhodium (III) | |
| Chen et al. | Determination of ammonia in water based on chemiluminescence resonance energy transfer between peroxymonocarbonate and branched NaYF4: Yb3+/Er3+ nanoparticles | |
| Tan et al. | Luminescence nucleotide/Eu3+ coordination polymer based on the inclusion of tetracycline | |
| CN106957243A (en) | A kind of copper ion detection probe based on aggregation-induced emission and its preparation method and application | |
| CN109971464B (en) | Preparation method of fluorescent probe for distinguishing peroxynitrite from hypochlorite based on xanthene and coumarin | |
| Zhang et al. | An unprecedented polyhydroxycarboxylic acid ligand bridged multi-Eu III incorporated tellurotungstate and its luminescence properties | |
| CN113340860A (en) | Manganese-doped carbon dot and Mn-CDs solution for detecting Fe3+, test paper, preparation method of test paper and detection method of test paper | |
| CN105837469B (en) | The preparation and application of a kind of long-chain p-nitrophenyl acylhydrazone gellike factor and its organic metal gel | |
| CN110194951B (en) | Tetraphenyl ethylene derivative fluorescent probe and preparation method thereof | |
| Yao et al. | Metal-ion-mediated synergistic coordination: construction of AIE-metallogel sensor arrays for anions and amino acids | |
| CN1257953C (en) | Rare earth transition mixed metal compounding material type zine ion fluorescent probe and its preparation method | |
| CN118222288A (en) | PtAg alloy nanocluster and preparation method and application thereof | |
| CN111892552A (en) | Triphenylamine derivative, preparation method thereof and application thereof in double-channel fluorescence detection of hydrogen sulfide | |
| CN112110955A (en) | AuCu with high phosphorescence quantum yield in air atmosphere14Nanocluster and method for preparing same | |
| CN102731479A (en) | Organic ligand, rare earth organic fluorescent probe material thereof and preparation method thereof | |
| CN102890076B (en) | The method of the twin-channel detection platinum ion of a kind of fluorescence-phosphorescence based on schiff bases | |
| CN110590784B (en) | Derivative based on pyrrolopyrroledione and preparation method and application thereof | |
| JP6573370B2 (en) | Method for producing platinum nanoparticle-containing composition and method for producing platinum nanoparticle | |
| CN111205344B (en) | Pure organic phosphorescent small-molecule material for methanol solvent recognition and preparation method thereof | |
| Yuan et al. | A luminescent Tb 3+ cholate hydrogel-based multi-functionalized platform for Hg 2+ and NO 2 detection | |
| CN111607100A (en) | Crystalline material based on iron-based porphyrin ligand, preparation and application thereof | |
| CN110698500A (en) | Gold alkynyl complex for silver ion sensing identification and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |