CN106248663B - A kind of active method based on colorimetric method or electrochemical process measurement protein kinase PKA - Google Patents
A kind of active method based on colorimetric method or electrochemical process measurement protein kinase PKA Download PDFInfo
- Publication number
- CN106248663B CN106248663B CN201610552292.9A CN201610552292A CN106248663B CN 106248663 B CN106248663 B CN 106248663B CN 201610552292 A CN201610552292 A CN 201610552292A CN 106248663 B CN106248663 B CN 106248663B
- Authority
- CN
- China
- Prior art keywords
- solution
- concentration
- pka
- agncs
- reaction
- 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.)
- Expired - Fee Related
Links
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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Plasma & Fusion (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Electrochemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The active method based on colorimetric method or electrochemical process measurement protein kinase PKA that the invention discloses a kind of, this method is with single stranded DNA (dC12) it is template and stabilizer, pass through NaBH4Restore AgNO3It synthesizes silver nanoclusters (AgNCs)；PKA catalytic hydrolysis of ATP utilizes Zr on the silk amino acid of γ transphorylation to peptide substrate4+The template DNA of the polypeptide of phosphorylation and AgNCs are combined, molecular probe is constructed；AgNCs can promote silver staining to react as crystal seed, and generating silver nano-grain makes solution become black from light yellow, inhibit silver staining to react after polypeptide package AgNCs, solution changes color speed slows down；Same AgNCs can generate electric signal in electrode surface, and silver staining reacts the electric signal for greatly strengthening AgNCs, and protein phosphorylation inhibits the generation of silver staining, and electric signal weakens；The activity that protein kinase PKA can be measured by colorimetric method or electrochemical process using the molecular probe, is had the advantages that easy to operate, high sensitivity and the detection used time is short.
Description
Technical field
The present invention relates to a kind of active method for measuring protein kinase PKA, in particular to a kind of building molecular probe, benefits
With colorimetric method or the method for electrochemical method determining protein kinase activity；Belong to biosensor technique field.
Background technique
Protein kinase catalytic protein phosphorylation is a kind of very important protein post-translational modification mode, can be adjusted thin
The function of most of albumen intracellular.The albumen of phosphorylation is closely related with numerous physiology courses, as signal transduction, metabolic regulation,
DNA damage reparation, genetic transcription and Apoptosis etc..Protein kinase is a kind of phosphotransferase, can be by atriphos
(ATP) γ-phosphate group on is transferred to the specific tyrosine of substrate protein (Tyr), threonine (Thr) and serine (Ser)
On residue.Protein kinase is the important member of enzyme family.Active normal protein kinase system is cellular signal transduction running
Core, but certain protein phosphorylations will be caused abnormal when its activity is abnormal or overexpression.The many diseases of the mankind just and
Protein phosphorylation is extremely closely related, such as cancer, diabetes and alzheimer.Organized since regulated protein kinases are each,
The life and death of each organ, even each cell, to have become the important drugs target of these complex diseases of early diagnosis and therapy
Point.Therefore, it measures the activity of protein kinase and then screens efficient kinases inhibitor in biomedical diagnostic and enzyme target
The extensive interest of people is evoked in the fields such as drug development.
People have been developed a large amount of detection active means of PKA, such as electrochemical process, fluorescence method, surface plasma
Resonance body method, labelled with radioisotope method and immunization etc., these detection means and method promote pair to a certain extent
Protein kinase activity research, however respectively there is certain limitation again.Radioactive element labelling technique, detection method is direct,
High sensitivity, but there are radioactive pollution, not can be carried out tissue mark；Immunization is more demanding to phosphorylation identification antibody,
Specificity is not strong, costly.Therefore, quick, simplicity, economy, high sensitivity are developed and without the inspection of the protein kinase activity of label
It surveys and high flux screening inhibits agent method to be very important.
Summary of the invention
In view of the deficienciess of the prior art, the purpose of the invention is to provide a kind of easy to operate, high sensitivity, at
This low and detection used time is short by colorimetric method or the active method of electrochemical method determining protein kinase PKA.
In order to solve the above technical problems, the present invention provides one kind to be based on the active side of colorimetric method for determining protein kinase PKA
Method, comprising the following steps:
1) using single stranded DNA containing cytimidine as template and stabilizer, with AgNO3For raw material and with NaBH4It is logical for reducing agent
Reduction method is crossed, generates and the AgNCs constituted on single stranded DNA containing cytimidine is supported on by nano silver dispersion；
2) by solution and ZrOCl containing AgNCs2Solution carries out hybrid reaction, obtains mixed liquor；
3) by Mg (NO3)2Solution, polypeptide solution, ATP solution and PKA solution carry out hybrid reaction, obtain enzyme reaction solution；
4) enzyme reaction solution and the mixed liquor are subjected to hybrid reaction to get molecular probe solution；
5) the molecular probe solution is mixed with silver staining liquid, is protected from light, and measures reaction product by ImageJ2x software
Corresponding average gray value；
6) using a series of PKA solution of various concentrations, 3), 4) He 5) step is repeated, is obtained a series of corresponding average
Gray value establishes the standard curve of PKA concentration Yu average gray value relationship；
7) Hela cell pyrolysis liquid to be measured is used, 3), 4) He 5) step is repeated and Hela to be measured is determined by standard curve
The concentration of PKA in cell pyrolysis liquid.
Preferred scheme is based on the active method of colorimetric method for determining protein kinase PKA, comprising the following steps:
It I is 0.8~1.2mM AgNO by 16~20 μ L concentration) under the conditions of being protected from light3Solution and 28~32 μ L concentration are 80
After~120 μM of DNA solutions mix 30~60min, 16~20 μ L concentration of addition are 0.8~1.2mM NaBH4Solution is gone back
Original reaction is to get AgNCs；
II) by 45~55 μ L concentration be 160~200 μM AgNCs solution and 8~12 μ L concentration be 0.8~1.2mM
ZrOCl2Solution mixing after shaking 1~2min, stands 15~25min of reaction, obtains mixed liquor；
III) Mg (the NO for being 80~120mM by 8~12 μ L concentration3)2, 8~12 μ L concentration be 23~27 μM polypeptide, 8~
The PKA mixing that the ATP solution and 8~12 μ L concentration that 12 μ L concentration are 40~60 μM are 0U/mL, under the conditions of 25~40 DEG C, instead
1~2h is answered, enzyme reaction solution is obtained；
IV) be added 30~50 μ L of enzyme reaction solution in the mixed liquor, shake 1~2min, stand reaction 15~
25min is to get molecular probe solution；
V the molecular probe solution is mixed with 80~120 μ L silver staining liquid), is protected from light, is surveyed by ImageJ2x software
Determine the corresponding average gray value of reaction product；
VI) be respectively using concentration 0.1,0.2,0.3,0.5U/mL PKA solution, repeat III), IV) and V) step, obtain
To a series of corresponding average gray values, the standard curve of PKA concentration and average gray value is established；
VII) use Hela cell pyrolysis liquid to be measured, repeat III), IV) and V) step is determined to be measured by standard curve
The concentration of PKA in Hela cell pyrolysis liquid.
More preferably scheme is based on the active method of colorimetric method for determining protein kinase PKA, comprising the following steps:
It I is 1mM AgNO by 18 μ L concentration) under the conditions of being protected from light3Solution and 30 μ L concentration are 100 μM of DNA solution mixing
After 30~60min, 18 μ L concentration of addition are 1mM NaBH4Solution carries out reduction reaction to get AgNCs；
II) by 50 μ L concentration be 180 μM AgNCs solution and 10 μ L concentration be 1mM ZrOCl2Solution mixing, concussion 1~
2min stands 15~25min of reaction, obtains mixed liquor；
III) Mg (the NO for being 100mM by 10 μ L concentration3)2, 10 μ L concentration be 25 μM polypeptide, 10 μ L concentration be 50 μM
The PKA that ATP solution and 10 μ L concentration are 0U/mL reacts 1~2h, obtains enzyme reaction solution under the conditions of 25~40 DEG C；
IV the 40 μ L of enzyme reaction solution) is added in the mixed liquor, 1~2min of concussion stands 15~25min of reaction, i.e.,
Obtain molecule probe solution；
V the molecular probe solution is mixed with 100 μ L silver staining liquid), is protected from light, is measured by ImageJ2x software anti-
Answer the corresponding average gray value of product；
VI) be respectively using concentration 0.1,0.2,0.3,0.5U/mL PKA solution, repeat III), IV) and V) step, obtain
To a series of corresponding average gray values, the standard curve of PKA concentration and average gray value is established；
VII) use Hela cell pyrolysis liquid to be measured, repeat III), IV) and V) step is determined to be measured by standard curve
The concentration of PKA in Hela cell pyrolysis liquid.
The present invention also provides one kind to measure the active method of protein kinase PKA based on electrochemical process, comprising the following steps:
A) using single stranded DNA containing cytimidine as template and stabilizer, with AgNO3For raw material and with NaBH4It is logical for reducing agent
Reduction method is crossed, generates and the AgNCs constituted on single stranded DNA containing cytimidine is supported on by nano silver dispersion；
B) by solution and ZrOCl containing AgNCs2Solution carries out hybrid reaction, obtains mixed liquor；
C) by Mg (NO3)2Solution, polypeptide solution, ATP solution and PKA solution carry out hybrid reaction, obtain enzyme reaction solution；
D) enzyme reaction solution and the mixed liquor are subjected to hybrid reaction to get molecular probe solution；
E) molecular probe solution is added dropwise after glassy carbon electrode surface, drying, is placed in silver staining liquid and impregnates, then passes through square wave
Voltammetry detection, obtains corresponding current signal value；
F) using a series of PKA solution of various concentrations, c), d) and e) step is repeated, a series of corresponding electric currents are obtained
Signal value establishes the standard curve of PKA concentration Yu current signal value relationship；
G) Hela cell pyrolysis liquid to be measured is used, c), d) and e) step is repeated and Hela to be measured is determined by standard curve
The concentration of PKA in cell pyrolysis liquid.
Preferred scheme measures the active method of protein kinase PKA based on electrochemical process, comprising the following steps:
It i) is 0.8~1.2mM AgNO by 16~20 μ L concentration under the conditions of being protected from light3Solution and 28~32 μ L concentration are 80
After~120 μM of DNA solutions are uniformly mixed 30~60min, 16~20 μ L concentration of addition are 0.8~1.2mM NaBH4Solution, into
Row reduction reaction is to get AgNCs；
Ii) by 23~27 μ L concentration be 160~200 μM AgNCs and 3~7 μ L concentration be 0.8~1.2mM ZrOCl2It is mixed
It closes, shakes 1~2min, stand 15~25min of reaction, obtain mixed liquor；
Iii) Mg (the NO for being 80~120mM by 3~7 μ L concentration3)2Solution, the polypeptide that 3~7 μ L concentration are 23~27 μM are molten
The PKA solution mixing that the ATP solution and 3~7 μ L concentration that liquid, 3~7 μ L concentration are 45~55 μM are 0U/mL, in 25~40 DEG C of items
Under part, 1~2h is reacted, enzyme reaction solution is obtained；
Iv) by enzyme reaction solution described in the mixed liquor and 18~22 μ L, 1~2min is shaken, stands 15~25min of reaction,
Up to molecular probe solution.
V) 7~8 μ L molecular probe solution are added dropwise and after drying, are placed at a temperature of 30~40 DEG C in glassy carbon electrode surface
2~4min is impregnated in silver staining liquid, using 1M KCl as electrolyte, within the scope of -0.15~0.25V, the frequency of 15HZ sweeps square wave
Volt-ampere curve obtains corresponding current signal value；
Vi) using concentration is respectively the PKA solution of 0.01,0.05,0.2,0.4,0.6,0.75,1U/mL, repeats iii),
Iv) and v) step, obtains a series of corresponding current signal values, establishes the standard curve of PKA concentration Yu current signal value relationship；
Vii) use Hela cell pyrolysis liquid to be measured, repeat iii), iv) and v) step is determined to be measured by standard curve
The concentration of PKA in Hela cell pyrolysis liquid.
More preferably scheme, based on electrochemical process measurement the active method of protein kinase PKA the following steps are included:
It i) is 1mM AgNO by 18 μ L concentration under the conditions of being protected from light3Solution and 30 μ L concentration are 100 μM of DNA solution mixing
After 30~60min, 18 μ L concentration of addition are 1mM NaBH4Solution carries out reduction reaction to get AgNCs；
Ii) by 25 μ L concentration be 180 μM AgNCs and 5 μ L concentration be 1mM ZrOCl21~2min is shaken in mixing, stands
15~25min is reacted, mixed liquor is obtained；
Iii) Mg (the NO for being 100mM by 5 μ L concentration3)2Polypeptide solution that solution, 5 μ L concentration are 25 μM, 5 μ L concentration are 50
μM ATP solution and 5 μ L concentration be 0U/mL PKA solution mix, under the conditions of 25~40 DEG C, react 1~2h, it is anti-to obtain enzyme
Answer liquid；
Iv) by enzyme reaction solution described in the mixed liquor and 20 μ L, shake 1~2min, stand 15~25min of reaction to get
Molecular probe solution.
V) 7.5 μ L molecular probe solution are added dropwise and after drying, are placed at a temperature of 30~40 DEG C in glassy carbon electrode surface
3min is impregnated in silver staining liquid, using 1M KCl as electrolyte, within the scope of -0.15~0.25V, the frequency of 15HZ sweeps square wave volt-ampere
Curve obtains corresponding current signal value；
Vi) using concentration is respectively the PKA solution of 0.01,0.05,0.2,0.4,0.6,0.75,1U/mL, repeats iii),
Iv) and v) step, obtains a series of corresponding current signal values, establishes the standard curve of PKA concentration Yu current signal value relationship；
Vii) use Hela cell pyrolysis liquid to be measured, repeat iii), iv) and v) step is determined to be measured by standard curve
The concentration of PKA in Hela cell pyrolysis liquid.
More preferably scheme, single-stranded DNA sequence containing cytimidine are CCC CCC CCC CCC.
More preferably scheme, silver staining liquid include AgNO3And hydroquinone.
It is of the invention based on the active method of colorimetric method for determining protein kinase PKA, comprising the following steps:
(1) AgNCs is synthesized: single stranded DNA (CCC CCC CCC CCC, dC to be rich in cytimidine12) simultaneously as template and
Stabilizer, NaBH4Restore AgNO3The AgNCs that synthesizing water-solubility is good, fluorescence intensity is stable；Firstly, with the revolving speed of 12000r/min
DNA is centrifuged 30~60min under the conditions of 4 DEG C, adds 127 μ L secondary waters to dilute in the DNA of 1OD, its concentration is made to reach 100 μ
M, then match 1mM AgNO with secondary water3, it is kept in dark place spare in 4 DEG C of refrigerators；Take 18 μ L 1mM AgNO3Solution and 30 μ L 100
μM DNA (wherein Ag+: DNA=6:1) be added in the brown centrifuge tube for filling 34 μ L secondary waters, at room temperature shake 30~
Then 18 μ L are newly prepared the NaBH of 1mM by 60min4(secondary water of ice is as solvent) is rapidly added above-mentioned DNA and AgNO3Mixing
In liquid, so that NaBH4Reduction Ag rapidly+For AgNCs；Final reaction solution is placed in 4 DEG C of refrigerators be kept in dark place it is 3~4 small
When, i.e. synthesis AgNCs；
(2) optimize the concentration of ATP: ATP is able to suppress silver staining reaction and occurs, and need to optimize to its concentration；In 96 orifice plates
Kong Zhongxian the AgNCs of the 50 above-mentioned synthesis of μ L is added, sequentially add the ATP of 50 μ L various concentrations from left to right, keep it final dense
Degree is 2.5,5,10,20,40 μM, finally adds 100 μ L silver staining liquid (silver staining liquid A: silver staining liquid B=1:1)；Observe blackening when
Between be respectively 5,15,20,30,40,90min, it is contemplated that test selected 5 μM of time restriction of process as optium concentration；
(3) activity of colorimetric method for determining protein kinase PKA, by 10 μ L 100mM Mg (NO3)2, 10 μ L, 25 μM of polypeptides, 10 μ
50 μM of ATP of L and 10 μ L ultimate densities are respectively 0,0.1,0.2,0.3, the PKA of 0.5U/mL in the centrifuge tube of 0.5mL,
1~2h of water-bath is under the conditions of 25~40 DEG C to get enzyme reaction solution；The AgNCs of the 50 above-mentioned synthesis of μ L is added in the Kong Zhongxian of 96 orifice plates,
Add 10 μ L 1mM ZrOCl2(Zr4+Ultimate density is 100 μM), 15~25min is reacted after shaking 1~2min, then take above-mentioned
40 μ L of enzyme is added in ELISA Plate, stands 15~25min of reaction after shaking 1~2min, is eventually adding 100 μ L silver staining liquid (silver staining liquid A
It is mixed with silver staining liquid B, ready-to-use), it is protected from light with masking foil sealing, photographs to record the degree of silver staining reaction every 1min,
The average gray value of fixed-area is measured by ImageJ2x software again；
Of the invention measures the active method of protein kinase PKA based on electrochemical process, comprising the following steps:
(1) electrode is handled: by the glass-carbon electrode of diameter 3mm in the Al containing 0.05 μm2O3Polishing cloth on polished,
Electrode after polishing is rinsed with secondary water, then is cleaned by ultrasonic respectively 3~5 minutes with dehydrated alcohol and secondary water, can will be attached to
The polishing powder of electrode surface remove it is clean, later with being dried with nitrogen；By the glass-carbon electrode handled well, Ag/AgCl reference electrode and
Platinum filament is put into jointly equipped with 5mmolL electrode-1Potassium ferricyanide solution reaction tank in, under -0.1~0.6V voltage, into
The scanning of row cyclic voltammetry, sets scanning speed as 0.1Vs-1, potential difference be less than 85mV, show that glassy carbon electrode surface is processed
Completely, be conducive to electrode modification；
(2) activity of electrochemical process measurement protein kinase PKA: by 5 μ L 100mM Mg (NO3)2, 5 μ L, 25 μM of polypeptides, 5 μ L
50 μM of ATP and 5 μ L ultimate densities are respectively 0,0.01,0.05,0.2,0.4,0.6,0.75, the PKA of 1U/mL in 0.5mL from
In heart pipe, 1~2h of water-bath is under the conditions of 25~40 DEG C to get enzyme reaction solution；The 25 above-mentioned conjunctions of μ L are added in the centrifuge tube of 0.5mL
At AgNCs, add 5 μ L 1mM ZrOCl2(Zr4+Ultimate density be 100 μM), shake 1~2min after stand reaction 15~
25min, then above-mentioned 20 μ L of enzyme is taken to be added in centrifuge tube, 15~25min of reaction is stood again after shaking 1~2min, obtains polypeptide packet
The AgNCs wrapped up in；Silver staining liquid A and silver staining liquid B are diluted 10 times, are divided in the centrifuge tube that two are protected from light；Take 7.5 μ L polypeptide packets
The AgNCs wrapped up in is added dropwise in the glassy carbon electrode surface handled well, in 30~40 DEG C of baking oven, about 40min drying；Take 30 μ L dilute
The electrode modified is immersed in silver staining liquid, takes out after 3min, knock out electricity in the centrifuge tube of 0.5mL by the silver staining liquid released
The remaining silver staining liquid of pole surface, the silver staining liquid for being sticked to surrounding them is wiped with lens wiping paper, using 1M KCl as electrolyte ,-
Within the scope of 0.15~0.25V, the frequency of 15HZ sweeps square wave volt-ampere curve.
Compared with the prior art, technology bring advantageous effects of the invention:
Technical solution of the present invention makes full use of PKA catalytic hydrolysis of ATP and can be by the γ transphorylation of ATP to polypeptide
Principle on the silk amino acid of substrate, with single stranded DNA (dC12) it is template and stabilizer, AgNCs is first prepared by reduction method, then
The polypeptide of phosphorylation is passed through into Zr4+It is combined together, is formed by the polypeptide package AgNCs's of phosphorylation with the template DNA of AgNCs
Molecular probe.The molecular probe has apparent advantage, can be used for the work of colorimetric method or electrochemical process measurement protein kinase PKA
Property.The molecular probe is wrapped in the surface AgNCs using the polypeptide of phosphorylation, inhibits silver staining reaction, by the solution colour depth, surveys
Gray value, size and the PKA activity of gray value are negatively correlated；Meanwhile AgNCs can generate electric signal in electrode surface, silver staining is anti-
AgNCs partial size should be promoted to become larger, so that signal value is significantly enhanced, the polypeptide of phosphorylation inhibits silver staining reaction to make electric signal
Enhancing degree slows down.
Colorimetric method of the invention does not need expensive instrument, easy to operate, and naked eye can direct observation experiment result.
Electrochemical process of the invention is simple, sensitive, quick, and mutually confirms with colorimetric method, highlights the method for the present invention
Reliability.
Detailed description of the invention
[Fig. 1] is the activity that the molecular probe that the present invention constructs measures protein kinase PKA for colorimetric method or electrochemical process
Schematic diagram.
[Fig. 2] illustrates that the polypeptide of phosphorylation is wrapped in the surface AgNCs histogram from charge angle for the present invention.
[Fig. 3] is the transmission electron microscope picture that AgNCs is synthesized in present example 1.
[Fig. 4] is ATP concentration optimization figure in present example 3, and ATP concentration is respectively 2.5,5,10,20,40 from left to right
μM。
[Fig. 5] is that the PKA enzyme reaction solution of various concentration and independent AgNCs are reacted with silver staining liquid in the embodiment of the present invention 5
Visualization figure when 25min.
[Fig. 6] be the embodiment of the present invention 5 in various concentration PKA relative gray values standard curve, detection range be 0.1~
0.5U/mL。
[Fig. 7] reacts 10min with silver staining liquid for various concentration medicine irritation Hela cell enzyme reaction liquid in present example 5
When visualization figure.
[Fig. 8] is the PKA electrochemical response signal graph of various concentration in the embodiment of the present invention 6.
[Fig. 9] is the PKA electrochemistry standard curve of various concentration in the embodiment of the present invention 6, detection range 0.01U/mL
~0.75U/mL.
[Figure 10] is that various concentration medicine irritation Hela cell enzyme reaction liquid reacts electrification with silver staining liquid in present example 6
Learn corresponding signal figure.
Specific embodiment
Following embodiment is intended to further illustrate the content of present invention, but the claims in the present invention protection scope be not limited to it is following
Embodiment.
Embodiment 1
To be rich in single stranded DNA (CCC CCC the CCC CCC, dC of cytimidine12) it is used as template and stabilizer, NaBH simultaneously4
Restore AgNO3The AgNCs that synthesizing water-solubility is good, fluorescence intensity is stable.Firstly, with the revolving speed of 12000r/min under the conditions of 4 DEG C
DNA is centrifuged 30~60min, adds 127 μ L secondary waters to dilute in the DNA of 1OD, so that its concentration is reached 100 μM, then use secondary water
With 1mM AgNO3, it is kept in dark place spare in 4 DEG C of refrigerators.Take 18 μ L 1mM AgNO3Solution and 30 μ L100 μM DNA (wherein Ag+: DNA=6:1) it is added in the brown centrifuge tube for filling 34 μ L secondary waters, 30~60min is shaken at room temperature, then by 18 μ
L newly prepares the NaBH of 1mM4(secondary water of ice is as solvent) is rapidly added above-mentioned DNA and AgNO3In mixed liquor, so that NaBH4
Reduction Ag rapidly+For AgNCs.Final reaction solution is placed in 4 DEG C of refrigerators, 3~4 hours are kept in dark place, is i.e. synthesis AgNCs.
Embodiment 2
By the glass-carbon electrode of diameter 3mm in the Al containing 0.05 μm2O3Polishing cloth on polished, the electrode after polishing
It is rinsed with secondary water, then is cleaned by ultrasonic respectively 3~5 minutes with dehydrated alcohol and secondary water, can will be attached to the throwing of electrode surface
Light powder remove it is clean, later with being dried with nitrogen.The glass-carbon electrode handled well, Ag/AgCl reference electrode and platinum filament are total to electrode
It is same to be put into equipped with 5mmolL-1Potassium ferricyanide solution reaction tank in, under -0.1~0.6V voltage, carry out cyclic voltammetry
Scanning, sets scanning speed as 0.1Vs-1, potential difference is less than 85mV, shows that glassy carbon electrode surface is processed clean, cover electricity
Polar cap is placed on spare in 4 DEG C of refrigerator.
Embodiment 3
ATP is able to suppress silver staining reaction and occurs, and need to optimize to its concentration.It is added on 50 μ L in the Kong Zhongxian of 96 orifice plates
The AgNCs of synthesis is stated, sequentially adds the ATP of 50 μ L various concentrations from left to right, makes 2.5,5,10,20,40 μ of its ultimate density
M finally adds 100 μ L silver staining liquid (silver staining liquid A: silver staining liquid B=1:1).The time of observation blackening is respectively 5,15,20,30,
40,90min, it is contemplated that selected 5 μM of time restriction for testing process are used as optium concentration.
Embodiment 4
Extract PKA in actual sample (Hela cell pyrolysis liquid): with containing fetal calf serum and 1640 dual anti-culture mediums,
36.5~37.5 DEG C, 4.5~5.5%CO2And Hela cell is cultivated in wet environment.With pancreas egg when cell is to logarithmic growth phase
White enzyme is digested, and is in suspension, with the forskolin (adenyl cyclase activator) and IBMX (ring of various concentration ratio
Adenylate phosphodiesterase inhibitors) it stimulates Hela cell (specific concentration is shown in table in Figure 10), it generates after 20~60min different
Concentration PKA.With cell crushing instrument by obtained Hela cell with the frequency of 20kHz, broken 30~60min at 0 DEG C, then with
10000~12000r/min is centrifuged 10~30min at 4 DEG C, removes organelle, and obtained supernatant is containing PKA's
Hela cell pyrolysis liquid.
Embodiment 5
By 10 μ L 100mM MgNO3, 10 μ L, 25 μM of polypeptides, 10 μ L, 50 μM of ATP and 10 μ L ultimate densities be respectively 0,
0.1,0.2,0.3, the PKA of 0.5U/mL is in the centrifuge tube of 0.5mL, and 1~2h of water-bath is anti-to get enzyme under the conditions of 25~40 DEG C
Answer liquid.The AgNCs of the 50 above-mentioned synthesis of μ L is added in the Kong Zhongxian of 96 orifice plates, adds 10 μ L 1mM ZrOCl2(Zr4+It is final dense
Degree is 100 μM), 15~25min is reacted after shaking 1~2min, then above-mentioned 40 μ L of enzyme reaction solution is taken to be added in ELISA Plate, concussion 1
15~25min of reaction is stood after~2min, is eventually adding 100 μ L silver staining liquid (silver staining liquid A and silver staining liquid B mixing, ready-to-use),
It is protected from light with masking foil sealing, photographs to record the degree of silver staining reaction every 1min, then solid by the measurement of ImageJ2x software
The average gray value for determining area is standard curve, R2=0.97856.
The active measurement of PKA in actual sample changes the PKA of various concentration into various concentration medicine irritation by the above operation
Hela cell pyrolysis liquid, it can be seen that as stimulating drug concentration increases, silver staining response inhabitation degree increased with it, and color becomes
Shallowly.
Embodiment 6
By 5 μ L 100mM Mg (NO3)2, 5 μ L, 25 μM of polypeptides, 5 μ L, 50 μM of ATP and 5 μ L ultimate densities be respectively 0,
0.01,0.05,0.2,0.4,0.6,0.75, the PKA of 1U/mL is in the centrifuge tube of 0.5mL, the water-bath 1 under the conditions of 25~40 DEG C
~2h is to get enzyme reaction solution.The AgNCs of the 25 above-mentioned synthesis of μ L is added in the centrifuge tube of 0.5mL, adds 5 μ L 1mM
ZrOCl2(Zr4+Ultimate density is 100 μM), 15~25min of reaction is stood after shaking 1~2min, then above-mentioned 20 μ L of enzyme is taken to be added
In centrifuge tube, 15~25min of reaction is stood again after shaking 1~2min, obtains the AgNCs of polypeptide package.By silver staining liquid A and silver staining
Liquid B dilutes 10 times, is divided in the centrifuge tube that two are protected from light.The AgNCs for taking 7.5 μ L polypeptides to wrap up is added dropwise in the glass handled well
Carbon electrodes, in 30~40 DEG C of baking oven, about 40min drying.The silver staining liquid for taking 30 μ L to dilute is in the centrifugation of 0.5mL
The electrode modified is immersed in silver staining liquid, takes out after 3min by Guan Zhong, knocks out the remaining silver staining liquid of electrode surface, with wiping mirror
Paper wipes the silver staining liquid for being sticked to surrounding them, using 1M KCl as electrolyte, within the scope of -0.15~0.25V, and the frequency of 15HZ
Square wave volt-ampere curve is swept, standard curve R is2=0.98697.
The active measurement of PKA in actual sample changes the PKA of various concentration into various concentration medicine irritation by the above operation
Hela cell pyrolysis liquid, it can be seen that as stimulating drug concentration increases, silver staining response inhabitation degree increased with it, electrochemistry
Signal strength gradually becomes smaller.
Basic principles and main features and advantages of the present invention of the invention have been shown and described above.The skill of the industry
Art personnel it should be appreciated that the present invention is not limited to the above embodiments, the above embodiments and description only describe
The principle of the present invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these
Changes and improvements all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and
Its equivalent thereof.
Claims (8)
1. one kind is based on the active method of colorimetric method for determining protein kinase PKA, it is characterised in that: the following steps are included:
1) using single stranded DNA containing cytimidine as template and stabilizer, with AgNO3For raw material and with NaBH4Pass through also for reducing agent
Former method generates and is supported on the AgNCs constituted on single stranded DNA containing cytimidine by nano silver dispersion；
2) by solution and ZrOCl containing AgNCs2Solution carries out hybrid reaction, obtains mixed liquor；
3) by Mg (NO3)2Solution, polypeptide solution, ATP solution and PKA solution carry out hybrid reaction, obtain enzyme reaction solution；
4) enzyme reaction solution and the mixed liquor are subjected to hybrid reaction to get molecular probe solution；
5) the molecular probe solution is mixed with silver staining liquid, is protected from light, and the phase of reaction product is measured by ImageJ2x software
Answer average gray value；
6) using a series of PKA solution of various concentrations, 3), 4) He 5) step is repeated, a series of corresponding average gray are obtained
Value, establishes the standard curve of PKA concentration Yu average gray value relationship；
7) Hela cell pyrolysis liquid to be measured is used, 3), 4) He 5) step is repeated and Hela cell to be measured is determined by standard curve
The concentration of PKA in lysate.
2. according to claim 1 be based on the active method of colorimetric method for determining protein kinase PKA, it is characterised in that: including
Following steps:
It I is 0.8~1.2mM AgNO by 16~20 μ L concentration) under the conditions of being protected from light3Solution and 28~32 μ L concentration are 80~120
After μM DNA solution mixes 30~60min, 16~20 μ L concentration of additions are 0.8~1.2mM NaBH4Solution restore anti-
It should be to get AgNCs；
II) by 45~55 μ L concentration be 160~200 μM AgNCs solution and 8~12 μ L concentration be 0.8~1.2mM ZrOCl2It is molten
Liquid mixing after shaking 1~2min, stands 15~25min of reaction, obtains mixed liquor；
III) Mg (the NO for being 80~120mM by 8~12 μ L concentration3)2, 8~12 μ L concentration be 23~27 μM polypeptide, 8~12 μ L
The PKA mixing that the ATP solution and 8~12 μ L concentration that concentration is 40~60 μM are 0U/mL, under the conditions of 25~40 DEG C, reaction 1~
2h obtains enzyme reaction solution；
IV 30~50 μ L of enzyme reaction solution) is added in the mixed liquor, shakes 1~2min, stands 15~25min of reaction,
Up to molecular probe solution；
V the molecular probe solution is mixed with 80~120 μ L silver staining liquid), is protected from light, is measured by ImageJ2x software anti-
Answer the corresponding average gray value of product；
VI) be respectively using concentration 0.1,0.2,0.3,0.5U/mL PKA solution, repeat III), IV) and V) step, obtain one
The corresponding average gray value of series, establishes the standard curve of PKA concentration and average gray value；
VII) use Hela cell pyrolysis liquid to be measured, repeat III), IV) and V) step by standard curve determines Hela to be measured
The concentration of PKA in cell pyrolysis liquid.
3. according to claim 1 or 2 be based on the active method of colorimetric method for determining protein kinase PKA, it is characterised in that: institute
The single-stranded DNA sequence containing cytimidine stated is CCC CCC CCC CCC.
4. according to claim 1 or 2 be based on the active method of colorimetric method for determining protein kinase PKA, it is characterised in that: institute
The silver staining liquid stated includes AgNO3And hydroquinone.
5. one kind measures the active method of protein kinase PKA based on electrochemical process, it is characterised in that: the following steps are included:
A) using single stranded DNA containing cytimidine as template and stabilizer, with AgNO3For raw material and with NaBH4Pass through also for reducing agent
Former method generates and is supported on the AgNCs constituted on single stranded DNA containing cytimidine by nano silver dispersion；
B) by solution and ZrOCl containing AgNCs2Solution carries out hybrid reaction, obtains mixed liquor；
C) by Mg (NO3)2Solution, polypeptide solution, ATP solution and PKA solution carry out hybrid reaction, obtain enzyme reaction solution；
D) enzyme reaction solution and the mixed liquor are subjected to hybrid reaction to get molecular probe solution；
E) molecular probe solution is added dropwise after glassy carbon electrode surface, drying, is placed in silver staining liquid and impregnates, then pass through square wave volt-ampere
Method detection, obtains corresponding current signal value；
F) using a series of PKA solution of various concentrations, c), d) and e) step is repeated, a series of corresponding current signals are obtained
Value, establishes the standard curve of PKA concentration Yu current signal value relationship；
G) Hela cell pyrolysis liquid to be measured is used, c), d) and e) step is repeated and Hela cell to be measured is determined by standard curve
The concentration of PKA in lysate.
6. according to claim 5 measure the active method of protein kinase PKA based on electrochemical process, it is characterised in that: packet
Include following steps:
It i) is 0.8~1.2mM AgNO by 16~20 μ L concentration under the conditions of being protected from light3Solution and 28~32 μ L concentration are 80~120
After μM DNA solution is uniformly mixed 30~60min, 16~20 μ L concentration of additions are 0.8~1.2mM NaBH4Solution is restored
Reaction is to get AgNCs；
Ii) by 23~27 μ L concentration be 160~200 μM AgNCs and 3~7 μ L concentration be 0.8~1.2mM ZrOCl2Mixing, shake
1~2min is swung, 15~25min of reaction is stood, obtains mixed liquor；
Iii) Mg (the NO for being 80~120mM by 3~7 μ L concentration3)2Solution, the polypeptide solution that 3~7 μ L concentration are 23~27 μM, 3
The PKA solution mixing that the ATP solution and 3~7 μ L concentration that~7 μ L concentration are 45~55 μM are 0U/mL, in 25~40 DEG C of conditions
Under, 1~2h is reacted, enzyme reaction solution is obtained；
Iv) by enzyme reaction solution described in the mixed liquor and 18~22 μ L, shake 1~2min, stand 15~25min of reaction to get
Molecular probe solution；
V) 7~8 μ L molecular probe solution are added dropwise and after drying, are placed in silver staining at a temperature of 30~40 DEG C in glassy carbon electrode surface
2~4min is impregnated in liquid, using 1M KCl as electrolyte, within the scope of -0.15~0.25V, the frequency of 15HZ sweeps square wave volt-ampere
Curve obtains corresponding current signal value；
Vi) using concentration be respectively 0.01,0.05,0.2,0.4,0.6,0.75,1U/mL PKA solution, repeat iii), iv) and
V) step obtains a series of corresponding current signal values, establishes the standard curve of PKA concentration Yu current signal value relationship；
Vii) use Hela cell pyrolysis liquid to be measured, repeat iii), iv) and v) step by standard curve determines Hela to be measured
The concentration of PKA in cell pyrolysis liquid.
7. according to claim 5 or 6 measure the active method of protein kinase PKA based on electrochemical process, it is characterised in that:
The single-stranded DNA sequence containing cytimidine is CCC CCC CCC CCC.
8. according to claim 5 or 6 measure the active method of protein kinase PKA based on electrochemical process, it is characterised in that:
The silver staining liquid includes AgNO3And hydroquinone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610552292.9A CN106248663B (en) | 2016-07-13 | 2016-07-13 | A kind of active method based on colorimetric method or electrochemical process measurement protein kinase PKA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610552292.9A CN106248663B (en) | 2016-07-13 | 2016-07-13 | A kind of active method based on colorimetric method or electrochemical process measurement protein kinase PKA |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106248663A CN106248663A (en) | 2016-12-21 |
| CN106248663B true CN106248663B (en) | 2019-07-16 |
Family
ID=57613926
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610552292.9A Expired - Fee Related CN106248663B (en) | 2016-07-13 | 2016-07-13 | A kind of active method based on colorimetric method or electrochemical process measurement protein kinase PKA |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106248663B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108760656B (en) * | 2018-05-23 | 2019-11-26 | 西南大学 | A kind of colorimetric method measuring adenosine kinase activity rapidly and sensitively |
| CN110987914B (en) * | 2019-11-19 | 2022-09-16 | 江苏大学 | Method for detecting and distinguishing phosphorylated protein based on Zr-MOF nanoenzyme and alpha-casein quantitative detection |
| CN111229193B (en) * | 2020-01-15 | 2022-10-18 | 重庆师范大学 | Application of zirconium dioxide nano particles as alkaline phosphatase nano mimics |
| CN111549096A (en) * | 2020-05-06 | 2020-08-18 | 浙江大学 | Method for detecting protein kinase A activity based on carbon nano material fluorescence |
| CN116027023B (en) * | 2022-08-15 | 2023-11-14 | 汉王科技股份有限公司 | Metal nanocluster, preparation method thereof and method for detecting olfactory receptor activation signal |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101358926A (en) * | 2008-09-12 | 2009-02-04 | 中国科学院长春应用化学研究所 | Method for unmarked colorimetric determination of enzyme based on argentum nanometer probe |
| CN103439482A (en) * | 2013-08-15 | 2013-12-11 | 中南大学 | Application of biosensing test paper based on N, N'-bis (trimethoxy silicyl propyl)-glutarimide |
| CN104849448A (en) * | 2015-05-12 | 2015-08-19 | 陕西师范大学 | Fluorescence quenching-based protein kinase activity analysis method |
| CN105548301A (en) * | 2016-01-28 | 2016-05-04 | 中南大学 | Method for measuring activity of protein kinase and method for measuring activity of protein kinase inhibitor |
-
2016
- 2016-07-13 CN CN201610552292.9A patent/CN106248663B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101358926A (en) * | 2008-09-12 | 2009-02-04 | 中国科学院长春应用化学研究所 | Method for unmarked colorimetric determination of enzyme based on argentum nanometer probe |
| CN103439482A (en) * | 2013-08-15 | 2013-12-11 | 中南大学 | Application of biosensing test paper based on N, N'-bis (trimethoxy silicyl propyl)-glutarimide |
| CN104849448A (en) * | 2015-05-12 | 2015-08-19 | 陕西师范大学 | Fluorescence quenching-based protein kinase activity analysis method |
| CN105548301A (en) * | 2016-01-28 | 2016-05-04 | 中南大学 | Method for measuring activity of protein kinase and method for measuring activity of protein kinase inhibitor |
Non-Patent Citations (4)
| Title |
|---|
| "A single electrochemical biosensor for detecting the activity and inhibition of both protein kinase and alkaline phosphatase based on phosphate ions induced deposition of redox precipitates";Congcong Shen et al.;《Biosensors and Bioelectronics》;20160507;第85卷;第220、221、223页 |
| "Silver Nanoclusters-Based Fluorescence Assay of Protein Kinase Activity and Inhibition";Congcong Shen et al.;《Analytical Chemistry》;20141217;第87卷(第1期);第693-698页 |
| "纳米材料的制备及其在蛋白激酶活性检测中的应用";王莹;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20150115(第01期);论文全文 |
| "蛋白激酶活性及抑制性检测研究进展";申聪聪、阳明辉;《化学传感器》;20150331;第35卷(第1期);第21-22页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106248663A (en) | 2016-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106248663B (en) | A kind of active method based on colorimetric method or electrochemical process measurement protein kinase PKA | |
| US11105767B1 (en) | Method for preparing dual-functional hybrid thin-film for self-calibration detection of tumor-derived exosomes | |
| Sun et al. | Nanoelectrochemistry of mammalian cells | |
| De León et al. | Three‐Dimensional (3D) cell culture monitoring: Opportunities and challenges for impedance spectroscopy | |
| Kenney et al. | A pH-sensing optode for mapping spatiotemporal gradients in 3D paper-based cell cultures | |
| Zhu et al. | 3D bimetallic Au/Pt nanoflowers decorated needle-type microelectrode for direct in situ monitoring of ATP secreted from living cells | |
| WO2008063151A2 (en) | Reaction sensing in living cells | |
| CN107132260B (en) | A kind of electrochemical sensor based on nano material detection Ractopamine | |
| Koukouviti et al. | 3D printed enzymatic microchip for multiplexed electrochemical biosensing | |
| Valiūnienė et al. | Investigation of active and inactivated yeast cells by scanning electrochemical impedance microscopy | |
| Gholivand et al. | Fabrication of an ultrasensitive impedimetric buprenorphine hydrochloride biosensor from computational and experimental angles | |
| Wang et al. | Monitoring of vesicular exocytosis from single cells using micrometer and nanometer-sized electrochemical sensors | |
| Bagby et al. | The button test: a small scale method using microdialysis cells for assessing protein solubility at concentrations suitable for NMR | |
| Yao et al. | A three-dimensional electrochemical biosensor integrated with hydrogel for cells culture and lactate release monitoring | |
| CN106093160B (en) | A kind of method of hydroxy apatite-base electrochemical probe construction method and measurement BACE1 activity and inhibition | |
| CN107153088A (en) | It is a kind of to be used to detect electrochemical sensor of tyrosine and its preparation method and application | |
| Chen et al. | Highly sensitive sandwich-type immunosensor with enhanced electrocatalytic durian-shaped MoS2/AuPtPd nanoparticles for human growth differentiation factor-15 detection | |
| Wu et al. | Site-selective probe for investigating the asynchronous unfolding of domains in bovine serum albumin | |
| CN105548301B (en) | A kind of method and the active method of kinases inhibitor measuring protein kinase activity | |
| Kintzios et al. | Development of a novel, multi-analyte biosensor system for assaying cell division: Identification of cell proliferation/death precursor events | |
| Lin et al. | Functional imaging-guided cell selection for evolving genetically encoded fluorescent indicators | |
| CN109580563A (en) | Nano sensor and preparation method and application thereof | |
| Tomida et al. | Stimulus-specific distinctions in spatial and temporal dynamics of stress-activated protein kinase kinase kinases revealed by a fluorescence resonance energy transfer biosensor | |
| Smith et al. | Kinetics of the potential-sensitive extrinsic probe oxonol VI in beef heart submitochondrial particles | |
| Shi et al. | The study of Nafion/xanthine oxidase/Au colloid chemically modified biosensor and its application in the determination of hypoxanthine in myocardial cells in vivo |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190716 Termination date: 20200713 |