CN105466876B - A kind of kit based on palladium nano-particles Visual retrieval Ismipur - Google Patents
A kind of kit based on palladium nano-particles Visual retrieval Ismipur Download PDFInfo
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- CN105466876B CN105466876B CN201511024536.8A CN201511024536A CN105466876B CN 105466876 B CN105466876 B CN 105466876B CN 201511024536 A CN201511024536 A CN 201511024536A CN 105466876 B CN105466876 B CN 105466876B
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- ismipur
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- peroxidase activity
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- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 title claims abstract description 88
- 229910052763 palladium Inorganic materials 0.000 title claims abstract description 44
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 39
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 title claims abstract description 30
- 230000000007 visual effect Effects 0.000 title claims abstract description 9
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 29
- 230000000694 effects Effects 0.000 claims abstract description 28
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 26
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 26
- 239000000243 solution Substances 0.000 claims abstract description 24
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 239000008055 phosphate buffer solution Substances 0.000 claims description 12
- XONPDZSGENTBNJ-UHFFFAOYSA-N molecular hydrogen;sodium Chemical compound [Na].[H][H] XONPDZSGENTBNJ-UHFFFAOYSA-N 0.000 claims description 7
- NJXQRPVPOUEUJS-UHFFFAOYSA-N 2-[3-amino-6-(4-amino-3,5-dimethylphenyl)-2,4-dimethylphenyl]ethanol Chemical compound CC1=C(C(=CC(=C1N)C)C1=CC(=C(N)C(=C1)C)C)CCO NJXQRPVPOUEUJS-UHFFFAOYSA-N 0.000 claims description 5
- 230000003139 buffering effect Effects 0.000 claims 1
- 210000002966 serum Anatomy 0.000 abstract description 9
- 210000002700 urine Anatomy 0.000 abstract description 9
- 238000001514 detection method Methods 0.000 abstract description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N EtOH Substances CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 7
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 abstract description 6
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 229940117820 purinethol Drugs 0.000 abstract 2
- 239000008363 phosphate buffer Substances 0.000 abstract 1
- 239000007864 aqueous solution Substances 0.000 description 13
- 238000002156 mixing Methods 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 10
- 238000000137 annealing Methods 0.000 description 8
- 239000012153 distilled water Substances 0.000 description 8
- 238000000034 method Methods 0.000 description 7
- 239000002253 acid Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000005549 deoxyribonucleoside Substances 0.000 description 5
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 4
- 239000011260 aqueous acid Substances 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 239000000460 chlorine Substances 0.000 description 4
- 229910052801 chlorine Inorganic materials 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 229910052708 sodium Inorganic materials 0.000 description 4
- 238000010792 warming Methods 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 3
- RJTANRZEWTUVMA-UHFFFAOYSA-N boron;n-methylmethanamine Chemical compound [B].CNC RJTANRZEWTUVMA-UHFFFAOYSA-N 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000004627 transmission electron microscopy Methods 0.000 description 3
- ZJWHIZQJEJGZCY-UHFFFAOYSA-N 4-(4-amino-3,5-dimethylphenyl)-2,6-dimethylaniline ethanol Chemical compound C(C)O.CC=1C=C(C=C(C1N)C)C1=CC(=C(N)C(=C1)C)C ZJWHIZQJEJGZCY-UHFFFAOYSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 238000001069 Raman spectroscopy Methods 0.000 description 2
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical compound C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003638 chemical reducing agent Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 238000002848 electrochemical method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000012279 sodium borohydride Substances 0.000 description 2
- 229910000033 sodium borohydride Inorganic materials 0.000 description 2
- DFIWJEVKLWMZBI-UHFFFAOYSA-M sodium;dihydrogen phosphate;phosphoric acid Chemical compound [Na+].OP(O)(O)=O.OP(O)([O-])=O DFIWJEVKLWMZBI-UHFFFAOYSA-M 0.000 description 2
- CMZYGFLOKOQMKF-UHFFFAOYSA-N 1-(3,5-dimethylphenyl)-3,5-dimethylbenzene Chemical group CC1=CC(C)=CC(C=2C=C(C)C=C(C)C=2)=C1 CMZYGFLOKOQMKF-UHFFFAOYSA-N 0.000 description 1
- 206010061623 Adverse drug reaction Diseases 0.000 description 1
- LXHQGXIAGLQVCC-UHFFFAOYSA-N C(C)O.CN(C1=CC=C(C2=CC=C(N(C)C)C=C2)C=C1)C Chemical compound C(C)O.CN(C1=CC=C(C2=CC=C(N(C)C)C=C2)C=C1)C LXHQGXIAGLQVCC-UHFFFAOYSA-N 0.000 description 1
- 208000006332 Choriocarcinoma Diseases 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 208000005125 Invasive Hydatidiform Mole Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- SUHGODOEKPYSKX-UHFFFAOYSA-M [O-]P(O)(O)=O.OP(O)(O)=O.OP(O)(O)=O.OP(O)(O)=O.[Na+].P Chemical compound [O-]P(O)(O)=O.OP(O)(O)=O.OP(O)(O)=O.OP(O)(O)=O.[Na+].P SUHGODOEKPYSKX-UHFFFAOYSA-M 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 229950006790 adenosine phosphate Drugs 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000001399 anti-metabolic effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000010290 biphenyl Nutrition 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- RQFCJASXJCIDSX-UUOKFMHZSA-N guanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 description 1
- 239000004226 guanylic acid Substances 0.000 description 1
- 235000013928 guanylic acid Nutrition 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229960001428 mercaptopurine Drugs 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- HKLZVEFFMGWYTE-UHFFFAOYSA-M potassium dihydrogen phosphate phosphoric acid Chemical compound P(=O)([O-])(O)O.P(=O)(O)(O)O.[K+].P(=O)(O)(O)O HKLZVEFFMGWYTE-UHFFFAOYSA-M 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- SPOMEWBVWWDQBC-UHFFFAOYSA-K tripotassium;dihydrogen phosphate;hydrogen phosphate Chemical compound [K+].[K+].[K+].OP(O)([O-])=O.OP([O-])([O-])=O SPOMEWBVWWDQBC-UHFFFAOYSA-K 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
Landscapes
- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of kit based on 6 purinethol of palladium nano-particles Visual retrieval, which includes：Palladium nano-particles liquid with peroxidase activity；3,3 ', 5,5 ' tetramethyl benzidine ethanol solutions；Aqueous hydrogen peroxide solution；PH is 4.0 sodium dihydrogen phosphate disodium hydrogen phosphate buffer solutions.It can be advantageously used in the measure of 6 purinethol contents in human serum and human urine using the kit of the present invention, with high sensitivity, selectivity is good, detection limit is low.
Description
Technical field
The invention belongs to nanometer technologies and bionics techniques field, relate particularly to a kind of based on palladium nano-particles visualization
Detect the kit of Ismipur.
Background technology
Ismipur, also known as mercaptopurine are purines antimetabolic type anticancer drug, in vivo by blocking hypoxanthine
It is changed into adenylic acid and guanylic acid and inhibits the synthesis of nucleic acid, is clinically mainly used for treating acute white blood
[Cancer, 1999,86 (6), 1080-1086] such as disease, chorioepithelioma and chorioadenomas.Taking Ismipur can produce
The raw side effects such as hepatic injury and bone marrow suppression.The activity of Ismipur is different due to individual difference and plasma concentration
[Biosens.Bioelectron.2013,41,844-847], clinic can be instructed by carrying out monitor drug concentration to medication patient
The rational use of medicines and for reduce adverse drug reaction be of great significance.Therefore, for human health, a kind of cheap, height is established
Sensibility, highly selective Ismipur detection method are most important.Up to the present, the detection method of Ismipur has
Electrochemical methods, high performance liquid chromatography, mass spectrometry, capillary electrophoresis, Raman spectroscopy, fluorescent spectrometry etc.
[Biosens.Bioelectron.2013,41,844–847；J.Electroanal.Chem.2012,665,63–69].At this
In a little methods, electrochemical methods are widely reported and many modified electrodes are used for the detection of Ismipur, but they
Poor repeatability and complicated electrode modification process limit their applications in actual sample；Although high performance liquid chromatography
Method, mass spectrometry, capillary electrophoresis and Raman spectroscopy detection Ismipur when have it is selective and sensitive, but it
Need expensive equipment and toxic solvent and often relate to complicated sample pretreatment；In for fluorescence detection, by
In no separation function cause selectivity it is relatively low, so its application it is limited [Biosens.Bioelectron.2013,41,
844–847]。
Invention content
It is a kind of based on palladium nano-particles Visual retrieval 6- mercaptos the purpose of the present invention is overcoming the deficiencies of the prior art and provide
The kit of base purine.
Technical scheme of the present invention is summarized as follows：
A kind of kit based on palladium nano-particles Visual retrieval Ismipur, including：
1) there is the palladium nano-particles liquid of peroxidase activity；
2) 3,3 ', 5,5 '-tetramethyl benzidine-ethanol solution；
3) aqueous hydrogen peroxide solution；
4) pH is 4.0 sodium dihydrogen phosphates-disodium hydrogen phosphate buffer solution.
The concentration of 3,3 ', 5,5 '-tetramethyl benzidine-ethanol solution is preferably 1-50mM.
The concentration of aqueous hydrogen peroxide solution is preferably 1-10M.
PH is that the concentration of 4.0 sodium dihydrogen phosphates-disodium hydrogen phosphate buffer solution is preferably 20-80mM.
Wherein, the palladium nano-particles with peroxidase activity are made of following methods：
1) annealing of deoxynucleotide：
By the deoxyribonucleoside aqueous acid of 2.5-250 μ L 2-200 μM, the phosphorus that 50-200 μ L 5-20mM pH are 3.0-9.0
Hydrochlorate buffer solution, which adds in the centrifuge tube of 1.5mL and adds in tri-distilled water, makes total volume for 400 μ L, mixing；It is warming up to 90-100
DEG C, 5-10min is maintained, is down to room temperature；
The sequence of deoxynucleotide is
Gb, wherein b=10-200；
Cc, wherein c=10-200；
(GC) d, wherein d=5-100；
(G2C) e, wherein e=4-70；
(G3C) f, wherein f=3-60；
(G4C) g, wherein g=2-50；
(GC2) h, wherein h=4-70；,
(GC3) i, wherein i=3-60；
(GC4) j, wherein j=2-50；
2) preparation of palladium nano-particles：
8-16 μ L 200-400 μM chlorine palladium acid sodium aqueous solutions are added to the deoxyribonucleoside sour water of the annealing of step 1) acquisition
In solution, the mixing at 20-30 DEG C stands 0.5-4h, adds in 6-24 μ L 0.6-2.4mM reducing agent aqueous solutions, mixing, in 20-
10-15h is stood at 30 DEG C, obtains the palladium nano-particles liquid with peroxidase activity.
Reducing agent is preferably sodium borohydride or dimethylamine borane.
Phosphate buffer solution used in the preparation of palladium nano-particles with peroxidase activity is preferably phosphorus
Acid dihydride sodium-disodium hydrogen phosphate buffer solution, potassium dihydrogen phosphate-dipotassium hydrogen phosphate cushioning liquid or sodium dihydrogen phosphate-phosphoric acid delay
Rush solution.
Advantages of the present invention：
It can be advantageously used in the measure of Ismipur content in human serum and human urine, tool using the kit of the present invention
There are high sensitivity, selective good, detection to limit low.
Description of the drawings
Fig. 1 is the transmission electron microscope picture of the palladium nano-particles with peroxidase activity synthesized in embodiment 1；
Fig. 2 is a concentration of 125mM of aqueous hydrogen peroxide solution in embodiment 1, and 3,3',5,5'-tetramethylbenzidine ethyl alcohol is molten
Liquid concentration is respectively the dynamic curve diagram of 0.015mM, 0.06mM, 0.08mM, 0.125mM, 0.165mM, 0.25mM；
Fig. 3 is the double reciprocal curve figure of Fig. 2.
Fig. 4 be in embodiment 1 3,3',5,5'-tetramethylbenzidine ethanol solution concentration be 0.125mM, aquae hydrogenii dioxidi
Solution concentration is respectively the dynamic curve diagram of 15mM, 30mM, 50mM, 75mM, 100mM, 125mM, 150mM；
Fig. 5 is the double reciprocal curve figure of Fig. 4.
Fig. 6 is the standard working curve figure of test example 1；
Specific embodiment
With reference to specific embodiment, the present invention is further illustrated.
The following examples are in order to enable those skilled in the art to more fully understand the present invention, but not to the present invention
It imposes any restrictions.
Embodiment 1
A kind of preparation method of the palladium nano-particles with peroxidase activity, includes the following steps：
(1) deoxynucleotide C10Annealing：
By 25 μ L, 20 μM of deoxynucleotide C10Aqueous solution, sodium dihydrogen phosphate-phosphoric acid hydrogen that 100 μ L 10mM pH are 5.0
Disodium buffer solution, which adds in the centrifuge tube of 1.5mL and adds in tri-distilled water, makes total volume for 400 μ L, mixing；95 DEG C are warming up to, dimension
5min is held, is down to room temperature；
(2) preparation of palladium nano-particles：
12 μ L, 300 μM of chlorine palladium acid sodium aqueous solutions are added to the deoxyribonucleoside aqueous acid of the annealing of step (1) acquisition
In, the mixing at 25 DEG C stands 2h, adds in 15 μ L 1.5mM dimethylamine borane aqueous solutions, and mixing stands 12h at 25 DEG C, obtains
To the palladium nano-particles liquid with peroxidase activity.It is 4.8nm to obtain average grain diameter with transmission electron microscopy.(see figure
1)
Detection：
Measure the peroxidase activity of palladium nano-particles：
In 6 4mL cuvettes, the palladium nano-particles with peroxidase activity of 9 μ L embodiments 1 acquisition are added in
Liquid, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution that 750 μ L 10mM pH are 4.0,38.2 μ L 0.125M aquae hydrogenii dioxidis
After solution, then sequentially add into 1-6 cuvette 0.015mM, 0.06mM of 15 μ L, 0.08mM, 0.125mM, 0.165mM,
0.25mM 3,3',5,5'-tetramethylbenzidine ethanol solutions, adding in tri-distilled water makes final volume for 3000 μ L, hybrid reaction
10min using the absorption spectrum at ultraviolet specrophotometer scanning 652nm, obtains absorbance, passes through Lineweaver-Burk
Equation calculation goes out its Michaelis constant.(see Fig. 2 and Fig. 3)
Palladium nano-particles with peroxidase activity show high enzymatic activity, to 3,3 ', 5,5 '-tetramethyl biphenyl
The Km values of amine are 0.051mM.
In 7 4mL cuvettes, the palladium nano-particles with peroxidase activity of 9 μ L embodiments 1 acquisition are added in
Liquid, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution that 750 μ L 10mM pH are 4.0,15 μ L 0.125mM 3,3 ', 5,5 '-
After tetramethyl benzidine ethanol solution, then sequentially add into 1-7 cuvette 15mM, 30mM of 38.2 μ L, 50mM, 75mM,
100mM, 125mM, 150mM aqueous hydrogen peroxide solution, adding in tri-distilled water makes final volume be used for 3000 μ L, hybrid reaction 10min
Absorption spectrum at ultraviolet specrophotometer scanning 652nm, obtains absorbance, is gone out by Lineweaver-Burk equation calculations
Its Michaelis constant.(see Fig. 4 and Fig. 5)
Palladium nano-particles with peroxidase activity show high enzymatic activity, and the Km values to hydrogen peroxide are
29.5mM。
Embodiment 2
A kind of preparation method of the palladium nano-particles with peroxidase activity, includes the following steps：
(1) deoxynucleotide G200Annealing：
By 2.5 μ L, 2 μM of deoxynucleotide G200Aqueous solution, potassium dihydrogen phosphate-phosphoric acid hydrogen that 50 μ L 5mM pH are 3.0
Dipotassium buffer solution, which adds in the centrifuge tube of 1.5mL and adds in tri-distilled water, makes total volume for 400 μ L, mixing；90 DEG C are warming up to, dimension
10min is held, is down to room temperature；
(2) preparation of palladium nano-particles：
8 μ L, 200 μM of chlorine palladium acid sodium aqueous solutions are added in the deoxyribonucleoside aqueous acid of the annealing of step (1) acquisition,
The mixing at 20 DEG C stands 4h, adds in 6 μ L 0.6mM sodium borohydride aqueous solutions, and mixing stands 15h at 20 DEG C, had
The palladium nano-particles liquid of peroxidase activity.
It is 1.0nm to obtain average grain diameter with transmission electron microscopy.Assay method is with embodiment 1, to 3,3 ', 5,5 '-tetramethyl
The Km values of base benzidine are 0.003mM, and the Km values to hydrogen peroxide are 481.4mM.
Embodiment 3
A kind of preparation method of the palladium nano-particles with peroxidase activity, includes the following steps：
(1) deoxynucleotide C200Annealing：
By 250 μ L, 200 μM of deoxynucleotide C200Aqueous solution, sodium dihydrogen phosphate-phosphorus that 200 μ L 20mM pH are 9.0
Acid solution, which adds in the centrifuge tube of 1.5mL and adds in tri-distilled water, makes total volume for 400 μ L, mixing；100 DEG C are warming up to, is maintained
5min is down to room temperature；
(2) preparation of palladium nano-particles：
16 μ L400 μM chlorine palladium acid sodium aqueous solutions are added in the deoxyribonucleoside aqueous acid of the annealing of step (1) acquisition,
The mixing at 30 DEG C stands 0.5h, adds in 24 μ L2.4mM dimethylamine borane aqueous solutions, and mixing stands 10h at 30 DEG C, obtains
Palladium nano-particles liquid with peroxidase activity.
It is 1.9nm to obtain average grain diameter with transmission electron microscopy.Assay method is with embodiment 1, to 3,3 ', 5,5 '-tetramethyl
The Km values of base benzidine are 0.014mM, and the Km values to hydrogen peroxide are 140mM.
Use G10、(GC)5、(GC)100、(G2C)4、(G2C)70、(G3C)3、(G3C)60、(G4C)2、(G4C)50、(GC2)4、
(GC2)70、(GC3)3、(GC3)60、(GC4)2、(GC4)50；C in alternative embodiment 1 respectively10, the other the same as in Example 1 obtained
The average grain diameters of the palladium nano-particles with peroxidase activity be shown in Table 1.And it is measured pair with the method identical with embodiment 1
The Km values of 3,3',5,5'-tetramethylbenzidine to the Km values of hydrogen peroxide, are shown in Table 1.
Table 1
Conclusion：Palladium nano-particles with peroxidase activity prepared by the method for the present invention are to the Km values of hydrogen peroxide
It is small, illustrate the affinity height of palladium nano-particles and hydrogen peroxide with peroxidase activity, reaction rate is fast, catalytic activity
It is high.
Embodiment 4
A kind of kit based on palladium nano-particles Visual retrieval Ismipur, including：
1) the palladium nano-particles liquid with peroxidase activity prepared by embodiment 1；
2) 3,3 ', 5,5 '-tetramethyl benzidines of 25mM-ethanol solution；
3) 9.8M aqueous hydrogen peroxide solutions；
4) 40mM pH are 4.0 sodium dihydrogen phosphates-disodium hydrogen phosphate buffer solution；
Test example 1
Ismipur is detected using the kit of embodiment 4：
In 11 4mL cuvettes, the palladium nanometer with peroxidase activity of 9 μ L embodiments 1 preparation is added in
Grain liquid, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution that 750 μ L 10mM pH are 4.0,38.2 μ L 0.125M hydrogen peroxide
After aqueous solution, 15 μ L 0.125mM 3,3',5,5'-tetramethylbenzidine-ethanol solution, then into 1-11 cuvette successively
Adding in 24 μ L concentration is respectively：0nM、2nM、100nM、150nM、200nM、250nM、300nM、350nM、500nM、600nM、
800nM Ismipur aqueous solutions, adding in tri-distilled water makes final volume be 3000 μ L, hybrid reaction 10min；(the concentration of each reagent
It is formed with the preparation of reagents of kit)；
Using ultraviolet specrophotometer scanning 652nm place absorption spectrum, and pass through digital camera photograph to record difference it is dense
The color of reaction solution, obtains color comparison picture in the presence of degree Ismipur；The Ismipur for calculating various concentration exists
Absorbance at 652nm obtains its calibration curve A652=1.1316-0.0025 [6-MP].(see Fig. 6) A652Absorption values with
6-MP concentration has good linear relationship in 0~350nM, and 3nM is limited to for Ismipur detection.
Test example 2
The content of the Ismipur in human urine sample and human serum sample is detected using the kit of embodiment 4：
In 8 4mL cuvettes, adding in 9 μ L has palladium nano-particles liquid, the 750 μ L of peroxidase activity
Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, 15 μ L 3,3 ', the 5,5 '-tetramethyl biphenyls of 0.125mM that 10mM pH are 4.0
After amine-ethanol solution, then it is separately added into the 4th cuvette of 1-：
The human serum of 10 times of 6 μ L dilutions,
6 μ L dilute 10 times human serum, 6 μ L 20nM6- purinethols,
6 μ L dilute 10 times human serum, 6 μ L 40nM6- purinethols,
6 μ L dilute 10 times of human serum, 6 μ L 80nM6- purinethols；
It is separately added into the 8th cuvette of 5-：
The human urine of 10 times of 6 μ L dilutions,
6 μ L dilute 10 times human urine, 6 μ L 20nM6- purinethols,
6 μ L dilute 10 times human urine, 6 μ L 40nM6- purinethols,
6 μ L dilute 10 times of human urine, 6 μ L 80nM6- purinethols；
It is added in again into each pipe：
38.2 μ L 125mM aqueous hydrogen peroxide solutions are added in, adding in tri-distilled water makes final volume for 3000 μ L, hybrid reaction
10min, using the absorption spectrum at ultraviolet specrophotometer scanning 652nm, according to linear equation determination sample solution, so as to obtain
Go out the content of Ismipur in sample solution.(being shown in Table 2)
Table 2
Embodiment 5
A kind of kit based on palladium nano-particles Visual retrieval Ismipur, including：
1) the palladium nano-particles liquid with peroxidase activity prepared by embodiment 2；
2) 3,3 ', 5,5 '-tetramethyl benzidines of 1mM-ethanol solution；
3) 1M aqueous hydrogen peroxide solutions；
4) 20mM pH are 4.0 sodium dihydrogen phosphates-disodium hydrogen phosphate buffer solution.
Assay method is the same as embodiment 4
It is demonstrated experimentally that the kit of the present embodiment can be used in the measure of Ismipur content in human serum and human urine.
Embodiment 6
A kind of kit based on palladium nano-particles Visual retrieval Ismipur, including：
1) the palladium nano-particles liquid with peroxidase activity prepared by embodiment 2；
2) 3,3 ', 5,5 '-tetramethyl benzidines of 50mM-ethanol solution；
3) 10M aqueous hydrogen peroxide solutions；
4) 80mM pH are 4.0 sodium dihydrogen phosphates-disodium hydrogen phosphate buffer solution.
Assay method is the same as embodiment 4
It is demonstrated experimentally that the kit of the present embodiment can be used in the measure of Ismipur content in human serum and human urine.
Claims (4)
1. a kind of kit based on palladium nano-particles Visual retrieval Ismipur, feature include：
1) there is the palladium nano-particles liquid of peroxidase activity；
2) 3,3 ', 5,5 '-tetramethyl benzidine-ethanol solution；
3) aqueous hydrogen peroxide solution；
4) pH is 4.0 sodium dihydrogen phosphates-disodium hydrogen phosphate buffer solution.
2. kit according to claim 1, it is characterized in that the 3,3',5,5'-tetramethylbenzidine-ethanol solution
A concentration of 1-50mM.
3. kit according to claim 1, it is characterized in that a concentration of 1-10M of the aqueous hydrogen peroxide solution.
4. kit according to claim 1, it is characterized in that the pH is 4.0 sodium dihydrogen phosphates-disodium hydrogen phosphate buffering
A concentration of 20-80mM of liquid.
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| CN110186862B (en) * | 2019-05-23 | 2021-08-03 | 天津大学 | Kit for visually detecting protamine based on palladium nano peroxidase |
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