CN107337699B - A kind of electrochemical luminescence probe tris (bipyridine) ruthenium-CBT and the preparation method and application thereof - Google Patents
A kind of electrochemical luminescence probe tris (bipyridine) ruthenium-CBT and the preparation method and application thereof Download PDFInfo
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- CN107337699B CN107337699B CN201710761437.0A CN201710761437A CN107337699B CN 107337699 B CN107337699 B CN 107337699B CN 201710761437 A CN201710761437 A CN 201710761437A CN 107337699 B CN107337699 B CN 107337699B
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- 238000004020 luminiscence type Methods 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical compound N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 title abstract description 58
- 239000007983 Tris buffer Substances 0.000 title abstract description 5
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- 239000000758 substrate Substances 0.000 claims abstract description 26
- 238000001514 detection method Methods 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 24
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 20
- BZSVVCFHMVMYCR-UHFFFAOYSA-N 2-pyridin-2-ylpyridine;ruthenium Chemical compound [Ru].N1=CC=CC=C1C1=CC=CC=N1.N1=CC=CC=C1C1=CC=CC=N1.N1=CC=CC=C1C1=CC=CC=N1 BZSVVCFHMVMYCR-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000009833 condensation Methods 0.000 claims abstract description 5
- -1 amino, carboxyl Chemical group 0.000 claims abstract description 4
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- YAYGSLOSTXKUBW-UHFFFAOYSA-N ruthenium(2+) Chemical compound [Ru+2] YAYGSLOSTXKUBW-UHFFFAOYSA-N 0.000 description 1
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- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0046—Ruthenium compounds
- C07F15/0053—Ruthenium compounds without a metal-carbon linkage
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/18—Metal complexes
- C09K2211/185—Metal complexes of the platinum group, i.e. Os, Ir, Pt, Ru, Rh or Pd
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
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Abstract
The present invention discloses a kind of electrochemical luminescence probe tris (bipyridine) ruthenium-CBT and the preparation method and application thereof, is related to technical field of biological.This method is that active amino is introduced on CABT, obtains active amino-CBT, for modifying to tris (bipyridine) ruthenium；Active amino-CBT is modified to Ru (bpy) by amino, carboxyl dehydration condensation3 2+On, obtain electrochemical luminescence probe Ru (bpy)3 2+-CBT.Method of the invention is simpler quickly, and versatility is good, the detection for different target proteins enzymes, by replacing corresponding peptide substrate sequence, so that it may realize the detection to different protease, have a wide range of application, it is good to the detection specificity of protease, high sensitivity.Probe design is simple, and operating procedure is brief, is easy in the fields promotion and application such as scientific research and clinical diagnosis；Inspection policies are simple, no special material modification and processing requirement.
Description
Technical field
The present invention relates to technical field of biological, in particular to a kind of electrochemical luminescence probe tris (bipyridine) ruthenium-CBT
(Ru(bpy)3 2+- CBT) and the preparation method and application thereof.
Background technique
Protease be one group can specific active site by protein or oligopeptides enzymatic hydrolysis at more fractionlet albumen water
Solve enzyme.Protease there are very rich, be almost present in all organisms, it is all that these enzymes almost participate in organism
Bioprocess, activity is closely related with many diseases, therefore protease is often used as target spot and the medical diagnosis on disease of drug therapy
Marker.The study found that protease plays key effect, including the life of protein digestibility, blood vessel in many physiology courses
The biological mistake such as transmitting at, blood clotting, wound repair, cell autophagy, aging, necrosis, Apoptosis and DNA hereditary information
Journey.Protease species are various, comprising trypsase, caspase family protein enzyme, fibrin ferment, matrix metalloproteinase (MMP),
Cathepsin etc. plays different effect in vivo.Many protease have become the life of disease forecasting and diagnosis
Therefore analyte detection target spot establishes a kind of highly sensitive and general proteinase activity detection method, basic scientific research and disease are faced
Bed diagnostic field is of great significance.
Currently, the method for more typical detection proteinase activity has fluorescence method, colorimetric method, electrochemical process, chemoluminescence method
With Electrochemiluminescince etc., fluorescence method application is more early and widely used, has developed quite mature, has constantly been based in recent years
Method report of the nano material probe of fluorescence method for real-time in-situ detection in the vitro detection and living cells of proteinase activity,
But such nano-probe is prepared with extremely strong technical and very high cost, seriously limits its clinical application.And colorimetric method
Sufficiently high sensitivity and stability cannot be reached by being disadvantageous in that, and be easy to be interfered by impurity and influence the accurate of detection
Degree.Electrochemical process has the advantages that sensitivity is good, accuracy is high and selectivity is good, but most highly sensitive detection platforms are mostly
Cumbersome electrode modification is carried out in early period, the technical requirements for such method for being are higher, and due to versatility deficiency, increase indirectly
The testing cost of such platform is added.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the primary purpose of the present invention is that providing a kind of electrochemical luminescence spy
Needle Ru (bpy)3 2+The preparation method of-CBT.
Another object of the present invention is to provide the electrochemical luminescence probe Ru being prepared by above-mentioned preparation method
(bpy)3 2+-CBT。
A further object of the present invention is to provide one kind to be based on electrochemical luminescence probe Ru (bpy)3 2+- CBT detects protease
Active method.
This method be a kind of electrochemical luminescence probe based on functionalization it is spontaneous and it is specific with cut by protease after
Peptide substrate group reaction cartridge, the platform of highly sensitive and high specific detection is carried out to protease.
The purpose of the invention is achieved by the following technical solution:
A kind of electrochemical luminescence probe Ru (bpy)3 2+The preparation method of-CBT, includes the following steps:
(1) active amino is introduced on CABT (6- amino -2- cyanobenzothiazole), obtains active amino-CBT, is used for
It modifies on tris (bipyridine) ruthenium；
(2) active amino-CBT is modified to Ru (bpy) by amino, carboxyl dehydration condensation3 2+On, obtain electrification
It learns luminescence probe Ru (bpy)3 2+-CBT。
CABT described in step (1) (6- amino -2- cyanobenzothiazole) is purchased from Shanghai OuKun Chemical Co., Ltd..
Active amino-CBT described in step (1) is preferably glycine-CBT；
The glycine-CBT synthesis step is as follows:
1) Boc-glycine (N- tertbutyloxycarbonyl-glycine) and NMM (N- methylmorpholine) is weighed to THF (tetrahydro furan
Mutter) in；
2) isobutyl chlorocarbonate is added under the conditions of 0 DEG C of nitrogen protection, reacts；
3) CABT is added into the reaction solution of step 2), after 0 DEG C is stirred to react, then is stirred to react at room temperature；
4) saturation NaHCO is added into the reaction solution of step 3)3, then be extracted with ethyl acetate, and use anhydrous sodium sulfate
Dry organic phase；
5) after removing organic phase by vacuum rotary steam, using silica gel column chromatography purified product, methylene chloride/methanol elution is eventually
Product Boc-glycine-CBT；
6) step 5) products therefrom is dissolved with methylene chloride, is added TFA (trifluoroacetic acid), reacts at room temperature；
7) purifying obtains glycine-CBT；
The mass ratio of Boc-glycine described in step 1) and NMM is 7:6；
The concentration of NMM described in step 1) is 7.5mg/mL；
The mass ratio of isobutyl chlorocarbonate described in step 2) and NMM are 2:3；
The condition of reaction described in step 2) is 0 DEG C of reaction 30min；
The mass ratio of CABT described in step 3) and Boc-glycine is 1:2；
0 DEG C of time being stirred to react described in step 3) is 2h；
The time being stirred to react at room temperature described in step 3) is 12h；
The volume ratio of methylene chloride and methanol is 10:1~5:1 in methylene chloride/methanol described in step 5)；
Final concentration of 20% of TFA described in step 6)；
The time reacted at room temperature described in step 6) is 12h；
Ru (bpy) described in step (2)3 2+From Ru (bpy)2dcbpy(PF6)2, Ru (bpy)2dcbpy(PF6)2Synthesis
Steps are as follows:
1) bis- (bipy 2,2' bipyridyl) rutheniums of 0.1g dichloro, 0.1g NaHCO are weighed3With 0.075g 4,4 '-dicarboxylic acids -2,2 ' -
Bipyridyl, 80 DEG C are stirred at reflux reaction 10h.
2) after ice bath 2h, with 1M sulphur acid for adjusting pH value to 4.4, filter paper is filtered to remove unreacted 4,4 '-dicarboxylic acids -2,
2 '-bipyridyls further rinse filter paper with 2~3mL methanol.
3) 6.25mL NaPF is added in Xiang Shangshu filtrate6Solution is stirred to react 2h at room temperature, with 1M sulphur acid for adjusting pH value
To 4, ice bath 4h.
4) 4 DEG C of 5000rpm are centrifuged 5min, and collection precipitates crystal, and lyophilization product obtains Ru (bpy)2dcbpy
(PF6)2。
5) 230mg DCC and 120mg NHS stirring and dissolving is weighed in 2mL anhydrous DMF, is subsequently placed on ice.
6) 190mg tris (bipyridine) ruthenium is added into said mixture, 30min is stirred to react in ice-water bath, it is anti-in room temperature
Answer 5h.
7) precipitating is discarded, Ru (bpy) is obtained in 5000g, 4 DEG C of centrifugation 5min2Dcbpy-NHS solution.
Active amino-CBT is modified to Ru (bpy) described in step (2)2dcbpy(PF6)2On reaction step and system
It is as follows:
A) 23.3mg active amino-CBT is taken to be slowly added into 1.5mL Ru (bpy)2In dcbpy-NHS solution, room temperature is stirred
Mix reaction 12h.
B) 100mL deionized water is added in Xiang Shangshu reaction system, extracts product with 10 × 100mL ethyl acetate, is used in combination
Anhydrous Na2SO4It is dry.
C) after removing organic phase by vacuum rotary steam, silica gel column chromatography purified product, methylene chloride/methanol (dichloro are utilized
Methane: methanol=5:1~1:1) elution final product Ru (bpy)3 2+-CBT。
A kind of electrochemical luminescence probe Ru (bpy)3 2+- CBT is prepared by above-mentioned preparation method.
The electrochemical luminescence probe Ru (bpy)3 2+Application of-the CBT in detection proteinase activity.
One kind being based on electrochemical luminescence probe Ru (bpy)3 2+The method that-CBT detects proteinase activity, includes the following steps:
(I) peptide substrate is incubated for altogether with protease sample to be checked, and protease inhibitors is then added and terminates endonuclease reaction, obtains
To proteolytic cleavage product CK-Biotin；
The peptide substrate sequence pattern is XXXXCK-Biotin, and XXXX is that protease specificity identifies sequence, polypeptide
Substrate can generate a dipeptides Cys-Lys-Biotin after being cut by protease；
(II) electrochemical luminescence probe Ru (bpy)3 2+- CBT and proteolytic cleavage product CK-Biotin group reaction cartridge, generation group
It fills compound Ru (bpy)3 2+-CBT-CK-Biotin；
(III) the coated enrichment with magnetic bead of Streptavidin, isolated complex Ru (bpy) are utilized3 2+-CBT-CK-Biotin；
(IV) Ru (bpy) isolated to step (III)3 2+- CBT-CK-Biotin carries out electrochemiluminescence analysis, from
And reflect the catalytic amount of protease indirectly.
Protease described in step (I) needs to have this feature: the sequence of its peptide substrate identified is special
, and allow to design a cysteine in restriction enzyme site one of carbon tip side.
Peptide substrate working concentration described in step (I) is preferably 1 μM.
Electrochemical luminescence probe Ru (bpy) described in step (II)3 2+- CBT and digestion products CK-Biotin group are anti-loaded
The condition answered is preferred are as follows: mild 15~60min of concussion reaction at room temperature；Preferably 30min.
Electrochemical luminescence probe Ru (bpy) described in step (II)3 2+- CBT working concentration is preferably 5 μM.
Assembling compound Ru (bpy) of the coated magnetic bead of Streptavidin described in step (III) to formation3 2+-CBT-
The enrichment of CK-Biotin and separation condition are preferred are as follows: after magnetic bead is added in digestion solution system, mild concussion is anti-at room temperature
15~30min (preferably 15min) is answered, is separated magnetic bead with solution with Magneto separate frame, each sample is mildly weighed with PBS respectively again
Outstanding washing 4 times, is finally resuspended magnetic bead with PBS.
Electrochemiluminescence analysis platform described in step (IV) is that Roche Elecsys2010 electrochemical luminescence is automatically exempted from
Epidemic disease analyzer and related matched reagent.
Basic principle of the invention is as shown in Figure 1:
During this investigation it turned out, we are anti-by the condensation between 2-cyanobenzothiazole (CBT) and cysteine for the first time
It should be applied to electrochemical luminescence hair detection proteinase activity.CBT-cysteine condensation reaction is initially found to occur in D- fluorescent
In the biosynthetic process of element, this reaction can be carried out quickly in physiological conditions, and second order reaction rate constant reaches
9.19M-1s-1.CBT generates new stable compound, CBT and the object for not containing 1,2- amineothiot after reacting with cysteine
Matter will not react, such as glutathione.Studies have shown that the peptide chain containing cysteine residues is not inside CBT and a kind of chain
It reacts, these all illustrate that CBT can only be reacted with the cysteine of peptide chain N-terminal.This kind of principle is applied to by we herein
In the detection of protease, such as Fig. 1 .A, by CBT modification to tris (bipyridine) ruthenium, designing being capable of half Guang of specific marker N-terminal
The electrochemical luminescence probe Ru (bpy) of propylhomoserin3 2+-CBT.Secondly, in the design of protease substrate polypeptide, in proteolytic cleavage position
The C-terminal side of point, introduces a cysteine, is marked with biotin in the C-terminal of peptide substrate, such a substrate exists
Cys-Lys-Biotin, Ru (bpy) can be generated after being cut by protease3 2+- CBT can specificity and Cys-Lys-Biotin
Group reaction cartridge, generates compound R u (bpy)3 2+The catalytic amount of-CBT-CK-Biotin, the amount protease of the compound are consistent.With
Streptavidin MagneSphere enrichment and separation above compound Ru (bpy)3 2+- CBT-CK-Biotin carries out electrochemiluminescence analysis,
What electrochemical luminescence intensity reflected is then the catalytic amount of protease, from detection proteinase activity (figure that can be accurate and special
1.B)。
The present invention compared with the existing technology, have following advantages and effects
(1) method of the invention is simpler quickly, and versatility is good, the detection for different target proteins enzymes, leads to
It crosses and replaces corresponding peptide substrate sequence, so that it may it realizes the detection to different protease, has a wide range of application, the inspection to protease
Survey specific good, high sensitivity.
(2) probe design is simple, and operating procedure is brief, is easy in the fields promotion and application such as scientific research and clinical diagnosis；
(3) inspection policies are simple, no special material modification and processing requirement.
Detailed description of the invention
Fig. 1 is the proteinase activity detection method schematic diagram based on electrochemical luminescence detection technique.
Fig. 2 is to electrochemical luminescence probe Ru (bpy)3 2+The Mass Spectrometer Method result of-CBT.
Fig. 3 is the verifying to this electrochemical luminescence detection of platform principle.
Fig. 4 is that this electrochemical luminescence platform that embodiment 1 obtains analyzes the detection sensitivity of trypsase.
Fig. 5 is that this electrochemical luminescence platform that embodiment 1 obtains analyzes the detection sensitivity of caspase-3.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The experimental method of specific experiment condition is not specified in the following example, usually according to conventional laboratory conditions or according to system
Make experiment condition proposed by manufacturer.
Ru used in embodiment (bpy)2dcbpy(PF6)2" Synthesis, labeling and in the literature
bioanalytical applications of a tris(2,2′-bipyridyl)ruthenium(II)-based
Electrochemiluminescence probe.2014 (9), 1146-1159 " are open.
Embodiment 1
A kind of electrochemical luminescence probe Ru (bpy)3 2+The preparation method of-CBT, specifically comprises the following steps:
(1) glycine-CBT is synthesized
Glycine-CBT is synthesized, to introduce active amino on CABT, for modifying to tris (bipyridine) ruthenium.
Specific step is as follows:
1. weighing in 175mg Boc-glycine and 150mg NMM to 20mL THF.
2. 100mg isobutyl chlorocarbonate, 0 DEG C of reaction 30min are added under the conditions of 0 DEG C of nitrogen protection.
3. 87.5mg CABT (being purchased from Shanghai OuKun Chemical Co., Ltd.) is added into above-mentioned reaction solution, 0 DEG C of stirring is anti-
After answering 2h, then it is stirred to react 12h at room temperature.
4. 100mL is added into above-mentioned reaction solution is saturated NaHCO3, then extracted with 3 × 150mL ethyl acetate, and use nothing
Aqueous sodium persulfate dries organic phase.
5. utilizing silica gel column chromatography purified product, methylene chloride: methanol (10:1 after removing organic phase by vacuum rotary steam
~5:1) elution final product Boc-glycine-CBT.
6. above-mentioned products therefrom is dissolved with methylene chloride, TFA is added to final concentration of 20%, reacts 12h at room temperature.
7. being purified by preparative high performance liquid chromatography and obtaining glycine-CBT.
(2) to Ru (bpy)3 2+Carry out glycine-CBT modification
Glycine-CBT is modified to Ru (bpy) by amino, carboxyl dehydration condensation3 2+On, obtain Ru
(bpy)3 2+-CBT。
Specific step is as follows:
The Ru (bpy)3 2+From Ru (bpy)2dcbpy(PF6)2, Ru (bpy)2dcbpy(PF6)2Synthesis step is as follows:
1. weighing bis- (bipy 2,2' bipyridyl) rutheniums of 0.1g dichloro, 0.1g NaHCO3With 0.075g 4,4 '-dicarboxylic acids -2,2 '
Bipyridyl, 80 DEG C are stirred at reflux reaction 10h.
2. after ice bath 2h, with 1M sulphur acid for adjusting pH value to 4.4, filter paper is filtered to remove unreacted 4,4 '-dicarboxylic acids -2,2 '
Bipyridyl is mended into one with 2~3mL methanol and rinses filter paper.
3. 6.25mL NaPF is added into above-mentioned filtrate6Solution is stirred to react 2h at room temperature, with 1M sulphur acid for adjusting pH value
To 4, ice bath 4h.
4.4 DEG C of 5000rpm are centrifuged 5min, and collection precipitates crystal, and lyophilization product obtains Ru (bpy)2dcbpy
(PF6)2。
5. weighing 230mg DCC and 120mg NHS stirring and dissolving in 2mL anhydrous DMF, it is subsequently placed on ice.
6. 190mg tris (bipyridine) ruthenium is added into said mixture, 30min is stirred to react in ice-water bath, it is anti-in room temperature
Answer 5h.
7. in 5000g, 4 DEG C of centrifugation 5min discard precipitating, obtain Ru (bpy)2Dcbpy-NHS solution.
Described modifies glycine-CBT to Ru (bpy)3 2+On reaction step and system it is as follows:
1. 23.3mg glycine-CBT is taken to be slowly added into 1.5mL Ru (bpy)2In dcbpy-NHS solution, room temperature is stirred
Mix reaction 12h.
2. 100mL deionized water is added into above-mentioned reaction system, product is extracted with 10 × 100mL ethyl acetate, is used in combination
Anhydrous Na2SO4It is dry.
3. utilizing silica gel column chromatography purified product, methylene chloride/methanol (dichloro after removing organic phase by vacuum rotary steam
Methane: methanol=5:1~1:1) elution final product Ru (bpy)3 2+-CBT。
Mass Spectrometer Method is carried out to obtained product, testing result is as shown in Fig. 2, show successfully to have prepared electrochemical luminescence
Probe Ru (bpy)3 2+-CBT。
Embodiment 2
The testing principle of electrochemical luminescence platform is verified
Using artificial synthesized CK-Biotin (Cys-Lys-Biotin) simulated albumin digestion products, probe is verified
Ru(bpy)3 2+The function of-CBT and the detection feasibility of platform.
Proof-Of Principle experimental procedure is as follows:
1. final concentration of 1 μM of CK-Biotin and 5 μM of Ru (bpy) is added in the PBS solution system of 100 μ L3 2+-
CBT probe, under room temperature concussion reaction 30min.
2. taking the 200 coated magnetic beads of μ L Streptavidin to be added in above-mentioned system, under room temperature mild concussion reaction
15min collects magnetic bead with Magneto separate frame, discards supernatant.After washing 4 times with 200 μ L PBS, then with 100 μ L PBS be resuspended magnetic bead,
Above-mentioned product is finally added to Roche Elecsys2010 electrochemical luminescence automatic lmunoassays analyzer and carries out photoelectric analysis, note
Record data result.
To the verification result of this electrochemical luminescence detection of platform principle, as shown in Figure 3, the results showed that electrochemical luminescence intensity
Enhance with the increase of CK-Biotin concentration, and (R in a linear relationship2=0.9925), illustrate the value of electrochemical luminescence intensity
It is able to reflect the amount of CK-Biotin in solution, therefore Ru (bpy)3 2+- CBT probe can be used for detecting protease and cut to substrate
It cuts.
Embodiment 3
1. the reaction time of trypsase and specific polypeptide substrate optimizes
Trypsase peptide substrate TRCK-Biotin (Thr-Arg-Cys-Lys-Biotin) purchased from Shanghai shine by force biology section
Skill Co., Ltd, trypsase, PMSF (phenylmethylsulfonyl fluoride) are purchased from sigma, and the coated magnetic bead of Streptavidin is purchased from Roche
Diagnosis.In the PBS solution system of 100 μ L, final concentration of 1 μM of substrate TRCK-Biotin and 1.5U mL is added-1Pancreas egg
White enzyme, be incubated for 1 in 37 DEG C of mild concussions respectively, 2,3,4,5,7, after 9min, the PMSF that 10 μM of 1 μ L is added terminates endonuclease reaction.
It is 50 μM of Ru (bpy) that 10 μ L concentration are added into above-mentioned system3 2+- CBT probe takes under room temperature after concussion reaction 30min
The 200 coated magnetic beads of μ L Streptavidin are added in above-mentioned system, under room temperature mild concussion reaction 15min, with magnetic point
Magnetic bead is collected from frame, is discarded supernatant.After washing 4 times with 200 μ L PBS, then with 100 μ LPBS be resuspended magnetic bead, finally by above-mentioned production
Object is added to Roche Elecsys2010 electrochemical luminescence automatic lmunoassays analyzer and carries out photoelectric analysis, records data result.It is excellent
The endonuclease reaction time after change is 5 minutes.
2. analysis of the electrochemical luminescence system to trypsase detection sensitivity
In the PBS solution system of 100 μ L, final concentration of 1 μM of substrate TRCK-Biotin is added and final concentration is respectively
0,5 × 10-4, 1 × 10-3, 5 × 10-3, 1 × 10-2, 5 × 10-2, 1 × 10-1, 5 × 10-1, 1,1.5,3U mL-1Trypsase,
After 37 DEG C of mild concussions are incubated for 5min, the PMSF that 10 μM of 1 μ L is added terminates endonuclease reaction.It is dense that 10 μ L are added into above-mentioned system
Degree is 50 μM of Ru (bpy)3 2+- CBT probe under room temperature after concussion reaction 30min, takes 200 μ L Streptavidin MagneSpheres to add
Enter into above-mentioned system, under room temperature mild concussion reaction 15min, collects magnetic bead with Magneto separate frame, discard supernatant.With 200 μ
After L PBS is washed 4 times, then with 100 μ L PBS magnetic bead is resuspended, it is electrochemical that above-mentioned product is finally added to Roche Elecsys2010
It learns the automatic lmunoassays analyzer that shines and carries out photoelectric analysis, and record data result.Testing result is as shown in figure 4, to tryptose
The detection of enzyme is 1 × 10-3~1.5U mL-1In the range of it is in a linear relationship, and according to formula ILOD=Icontrol+3Istdev(con)
It calculates to detect and is limited to 8.4 × 10-4U mL-1。
Embodiment 4
The reaction time of 1.caspase-3 protease and specific polypeptide substrate optimizes
Caspase-3 peptide substrate DEVDCK-Biotin (Asp-Glu-Val-Asp-Cys-Lys-Biotin) is purchased from upper
Hai Qiangyao Biotechnology Co., Ltd, caspase-3 protease are purchased from BioVision Incorporated, caspase-3 egg
White enzyme inhibitor Ac-DEVD-CHO is purchased from green skies biotechnology.In the PBS solution system of 100 μ L, final concentration of 1 μ is added
Substrate DEVDCK-Biotin and the 0.0025U mL of M-1Caspase-3 protease, be incubated for 1 in 37 DEG C of mild concussions respectively, 2,
3, after 4,5,6,7 and 8h, the Ac-DEVD-CHO that 1 μM of 1 μ L is added terminates endonuclease reaction.Ru is carried out according to the method for embodiment 3
(bpy)3 2+The group reaction cartridge of-CBT probe is separated and is detected with subsequent.It is analyzed according to the ECL data of output optimal anti-
It is 6h between seasonable.
2. analysis of pair this electrochemical luminescence system to caspase-3 protease detection sensitivity
In the PBS solution system of 100 μ L, final concentration of 1 μM of substrate DEVDCK-Biotin and final concentration difference is added
It is 0,2.5 × 10-6, 5 × 10-6, 7.5 × 10-6, 1 × 10-5, 5 × 10-5, 7.5 × 10-5, 1 × 10-4, 5 × 10-4, 1 × 10-3,
1.5×10-3, 2 × 10-3, 2.5 × 10-3, 3 × 10-3, 3.5 × 10-3U mL-1Caspase-3 protease, 37 DEG C of mild concussions
After being incubated for 6h, the Ac-DEVD-CHO that 1 μM of 1 μ L is added terminates endonuclease reaction.Ru (bpy) is carried out according to the method for embodiment 33 2+-
The group reaction cartridge of CBT probe is separated and is detected with subsequent.Testing result is as shown in figure 5, detection to caspase-3 protease
5 × 10-6~1.5 × 10-3U mL-1In the range of it is in a linear relationship, and according to formula ILOD=Icontrol+3Istdev(con)It calculates
It must detect and be limited to 5 × 10-6U mL-1。
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>South China Normal University
<120>a kind of electrochemical luminescence probe tris (bipyridine) ruthenium-CBT and the preparation method and application thereof
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223>trypsase peptide substrate
<220>
<221>Biotin is modified
<222> (4)..(4)
<400> 1
Thr Arg Cys Lys
1
<210> 2
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223>caspase-3 peptide substrate
<220>
<221>Biotin is modified
<222> (6)..(6)
<400> 2
Asp Glu Val Asp Cys Lys
1 5
Claims (8)
1. a kind of electrochemical luminescence probe Ru (bpy)3 2+The preparation method of-CBT, it is characterised in that include the following steps:
(1) active amino is introduced on CABT, obtains active amino-CBT, for modifying to tris (bipyridine) ruthenium；
(2) active amino-CBT is modified to Ru (bpy) by amino, carboxyl dehydration condensation3 2+On, obtain electrochemistry hair
Light probe Ru (bpy)3 2+-CBT；
Active amino-CBT described in step (1) is glycine-CBT；
The structure of the glycine-CBT is as follows:
Ru (bpy) described in step (2)3 2+From Ru (bpy)2dcbpy(PF6)2；
The Ru (bpy)2dcbpy(PF6)2Structure it is as follows:
The Ru (bpy)3 2+The structure of-CBT is as follows:
2. a kind of electrochemical luminescence probe Ru (bpy)3 2+- CBT, which is characterized in that the Ru (bpy)3 2+The structure of-CBT is such as
Shown in lower:
3. electrochemical luminescence probe Ru (bpy) as claimed in claim 23 2+Application of-the CBT in detection proteinase activity.
4. one kind is based on electrochemical luminescence probe Ru (bpy)3 2+The method of-CBT detection proteinase activity, it is characterised in that including
Following steps:
(I) peptide substrate is incubated for altogether with protease sample to be checked, and protease inhibitors is then added and terminates endonuclease reaction, obtains egg
White digestion products CK-Biotin；
The peptide substrate sequence pattern is XXXXCK-Biotin, and XXXX is that protease specificity identifies sequence, peptide substrate
A dipeptides Cys-Lys-Biotin can be generated after being cut by protease；
(II) electrochemical luminescence probe Ru (bpy) as claimed in claim 23 2+- CBT and proteolytic cleavage product CK-Biotin is assembled
Reaction generates assembling compound Ru (bpy)3 2+-CBT-CK-Biotin；
(III) the coated enrichment with magnetic bead of Streptavidin, isolated complex Ru (bpy) are utilized3 2+-CBT-CK-Biotin；
(IV) Ru (bpy) isolated to step (III)3 2+- CBT-CK-Biotin carries out electrochemiluminescence analysis, thus
The reversed catalytic amount for reflecting protease；
It is described based on electrochemical luminescence probe Ru (bpy)3 2+The route that-CBT detects the method for proteinase activity is as follows:
5. according to claim 4 be based on electrochemical luminescence probe Ru (bpy)3 2+The method that-CBT detects proteinase activity,
It is characterized by:
Electrochemical luminescence probe Ru (bpy) described in step (II)3 2+- CBT and digestion products CK-Biotin group reaction cartridge
Condition are as follows: mild 15~60min of concussion reaction at room temperature.
6. according to claim 4 be based on electrochemical luminescence probe Ru (bpy)3 2+The method that-CBT detects proteinase activity,
It is characterized by:
Peptide substrate working concentration described in step (I) is 1 μM；
Electrochemical luminescence probe Ru (bpy) described in step (II)3 2+- CBT working concentration is 5 μM.
7. according to claim 4 be based on electrochemical luminescence probe Ru (bpy)3 2+The method that-CBT detects proteinase activity,
It is characterized by:
Assembling compound Ru (bpy) of the coated magnetic bead of Streptavidin described in step (III) to formation3 2+-CBT-CK-
The enrichment and separation condition of Biotin are as follows: after magnetic bead is added in digestion solution system, at room temperature mild concussion reaction 15~
30min is separated magnetic bead with solution with Magneto separate frame, and washing 4 times is mildly resuspended in each sample with PBS respectively again, finally uses PBS
Magnetic bead is resuspended.
8. according to claim 4 be based on electrochemical luminescence probe Ru (bpy)3 2+The method that-CBT detects proteinase activity,
It is characterized by:
Electrochemiluminescence analysis platform described in step (IV) is automatically immune point of Roche Elecsys2010 electrochemical luminescence
Analyzer and related matched reagent.
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