CN106770517A - A kind of method of mice skeletal myostatin content after detection exhausted movemeat - Google Patents
A kind of method of mice skeletal myostatin content after detection exhausted movemeat Download PDFInfo
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- CN106770517A CN106770517A CN201611166543.6A CN201611166543A CN106770517A CN 106770517 A CN106770517 A CN 106770517A CN 201611166543 A CN201611166543 A CN 201611166543A CN 106770517 A CN106770517 A CN 106770517A
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- 108010056852 Myostatin Proteins 0.000 title claims abstract description 60
- 241000699670 Mus sp. Species 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 17
- 102000004472 Myostatin Human genes 0.000 title claims abstract 20
- 238000001514 detection method Methods 0.000 title claims description 16
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims abstract description 66
- 239000002245 particle Substances 0.000 claims abstract description 45
- 229910052697 platinum Inorganic materials 0.000 claims abstract description 33
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000010931 gold Substances 0.000 claims abstract description 22
- 229910052737 gold Inorganic materials 0.000 claims abstract description 22
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- GPRSOIDYHMXAGW-UHFFFAOYSA-N cyclopenta-1,3-diene cyclopentanecarboxylic acid iron Chemical compound [CH-]1[CH-][CH-][C-]([CH-]1)C(=O)O.[CH-]1C=CC=C1.[Fe] GPRSOIDYHMXAGW-UHFFFAOYSA-N 0.000 claims abstract description 7
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- 150000004062 1,4-benzoquinone imines Chemical class 0.000 claims description 2
- NGNBDVOYPDDBFK-UHFFFAOYSA-N 2-[2,4-di(pentan-2-yl)phenoxy]acetyl chloride Chemical compound CCCC(C)C1=CC=C(OCC(Cl)=O)C(C(C)CCC)=C1 NGNBDVOYPDDBFK-UHFFFAOYSA-N 0.000 claims description 2
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Classifications
-
- 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
-
- 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
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Abstract
The invention belongs to electrochemical sensor field, its principle is that the effect of antibody and golden nanometer particle is recycled using golden nanometer particle modified electrode, prepares the gold electrode of antibody modification, obtains final product antibody/golden nanometer particle/gold electrode.Then by antibody/golden nanometer particle/gold electrode sample of the immersion containing myostatin, antibody modification nano platinum particle is added.Sandwich structure is formed using the effect of myostatin and antibody, in p-nitrophenol PNP, NaBH4, ferrocenecarboxylic acid Fc system in there is redox cycle, electrochemical response signal is amplified.Power according to electrochemical signals can realize the measure to myostatin；And mice skeletal myostatin content after exhausted movemeat is determined, method have it is simple to operate, it is with low cost, the characteristics of sensitivity is high.
Description
Technical field
The invention belongs to electrochemical sensor field, and in particular to mice skeletal muscle life after one kind detection exhausted movemeat
The method for suppressing cellulose content long.
Background technology
Myostatin (Myostatin, MSTN), is Skeletal Muscle Growth also known as Growth differentiation factor 8 (GDF-8)
The major regulator of development, it is mainly expressed in skeletal muscle, and it develops inhibited to Skeletal Muscle Growth, is in recent years
Come a kind of muscle specific albumen [Furihata T, Kinugawa S, Fukushima A, the et al.Serum for finding
myostatin levels are independently associated with skeletal muscle wasting in
patients with heart failure[J].International Journal of Cardiology,2016,22:
483-487].Myostatin content in the tissue is grown with important influence, therefore to muscle growth to flesh
The measure for suppressing cellulose content has great importance.The method that document report determines myostatin mainly has fluorescence method
[Tian Zhenjun, He Zhixiong, Liu Zhiwei wait persistently and interval aerobic exercise are to heart infarction rat heart muscle Myostatin and its by body surface
Influence [J] the sports sciences for reaching, 2013,33 (11):66-74.], chemoluminescence method [Chang H M, Fang L, Cheng J
C,et al.Growth differentiation factor 8down-regulates pentraxin 3in human
granulosa cells[J].Molecular&Cellular Endocrinology,2015,404:82-90.Chang H M,
Pan H H,Cheng J C,et al.Growth differentiation factor 8suppresses cell
proliferation by up-regulating CTGF expression in human granulosa cells.[J]
.Molecular&Cellular Endocrinology,2016,422:9-17.].In recent years, urged based on electrochemical redox
Changing reaction assay new method becomes one of focus [Das J, Aziz M A, Yang H.A nanocatalyst-based
assay for proteins:DNA-free ultrasensitive electrochemical detection using
catalytic reduction of p-nitrophenol by gold-nanoparticle labels.[J].Journal
of the American Chemical Society,2006,128(128):16022-16023.Hun X,Xie G,Luo
X.Scaling up an electrochemical signal with a catalytic hairpin assembly
coupling nanocatalyst label for DNA detection.[J].Chemical Communications,
2015,51(33):7100-7103].In order to further improve the sensitivity to MSTN detections, nano platinum particle labelled antibody is used,
With the catalytic amplification of nano platinum particle, a kind of electrochemistry new method of highly sensitive detection myostatin is constructed,
And for the measure of mice skeletal myostatin content after exhausted movemeat.
The content of the invention
It is contemplated that it is simple to operate to invent a kind of method, with low cost, sensitivity measure myostatin high
Method.
Realizing goal of the invention technical scheme is：
A kind of method of mice skeletal myostatin content after detection exhausted movemeat.Its principle is to utilize Jenner
Rice corpuscles modified electrode, recycles the effect of antibody and golden nanometer particle, prepares the gold electrode of antibody modification, obtains final product antibody/gold
Nano-particle/gold electrode.Then antibody/golden nanometer particle/gold electrode is immersed in the sample containing myostatin, then
Add antibody modification nano platinum particle.Sandwich structure is formed using the effect of myostatin and antibody, right
Nitrophenol PNP, NaBH4, ferrocenecarboxylic acid Fc system in, nano platinum particle is in NaBH4It is right that the lower catalysis PNP of effect is converted into
Amino-phenol PAP, the PAP of generation is by the Fc oxidations in solution, oxidation product 1,4-benzoquinone imines p-quinone imine and quilt
NaBH4PAP is reduced into, lasting redox is produced between such PAP and oxidation product, so that electrochemical signals are produced,
There is redox cycle in the presence of nano platinum particle, electrochemical response signal is amplified.According to the strong of electrochemical signals
The weak measure that can be realized to myostatin；And mice skeletal myostatin content after exhausted movemeat is entered
Measure is gone.
Determination step is：
(1) preparation of nano platinum particle
First, the glass apparatus chloroazotic acid foam washing used by nano platinum particle, dry for standby will be prepared.Then by the dense of 7mL
The ascorbic acid for adding that 1.0mL concentration is 1% is spent in the platinum acid chloride solution for 1%, under the conditions of 80 DEG C, is stirred 20 minutes, when molten
Liquid becomes dark brown and stops heating, obtains nano platinum particle, is cooled to room temperature, is saved backup under the conditions of 4 DEG C.
(2) preparation of antibody modification nano platinum particle
First, the 2mL nano platinum particles solution for preparing is mixed with the phosphate buffer solution of 8mL pH 7.4, is diluted
Nano platinum particle, nano platinum particle 2mL and 0.5mL the myostatin antibody for then pipetting dilution mixes, 37 DEG C of bars
Under part, concussion reaction 24h obtains antibody/nano platinum particle compound, using supercentrifuge eccentric cleaning after, be dispersed again in
In PBS cushioning liquid, preserved at 4 DEG C.
(3) preparation of antibody modification gold electrode
First, the solution of gold nanoparticles of 10 μ L is added dropwise in the gold electrode surfaces handled well, 10h is incubated under the conditions of 4 DEG C,
Obtain golden nanometer particle modified gold electrode.Then by the myostatin antibody drop coating of 10 μ L in golden nanometer particle modification gold electricity
Pole surface, under the conditions of 37 DEG C, react 24h, obtain antibody/golden nanometer particle/gold electrode, with phosphate buffer solution cleaning electrode after,
Preserved at 4 DEG C.
(4) detection of myostatin
Antibody/golden nanometer particle/gold electrode is immersed in the sample solution containing myostatin, under the conditions of 37 DEG C
Reaction 2h, then electrode is taken out, flushed three times with phosphate buffer solution, be inserted into pH 7.4 PNP containing 5mmol/L,
5mmol/L NaBH4In the phosphate buffer solution of 5mmol/L Fc, on electrochemical workstation, electricity is detected with cyclic voltammetry
Chemical signal, current potential is -200~600mV (vs.SCE), uses 50mV s-1Velocity scanning.
(5) mice skeletal myostatin content detection after exhausted movemeat
Exhausted movemeat is carried out to mouse and is drawn materials, mice skeletal myostatin contains after detection exhausted movemeat
Amount, as a result shows, the content of mice skeletal myostatin is 4.56ng/mg, control group mice bone after exhausted movemeat
The content of bone flesh myostatin is 5.72ng/mg；Show, mice skeletal myostatin after exhausted movemeat
Content decreases compared with control group.
Described myostatin, myostatin antibody are purchased from R＆D Systems companies.
Described chloroplatinic acid (H2PtCl6), ascorbic acid, ferrocenecarboxylic acid (Fc) purchased from Chinese medicines group chemical reagent it is limited
Company.
Described sodium borohydride (NaBH4), p-nitrophenol (PNP) be purchased from Tianjin chemical reagent Co., Ltd.
The water used in experimentation is redistilled water.
By the KH of 21mL 0.2mol/L2PO4, 78mL 0.2mol/L Na2HPO4·12H2O mixes, and adds KCl to consolidate
Body so that the concentration of KCl is 0.1mol/L, obtains the PBS cushioning liquid that 0.1mol/L pH are 7.4.
Described phosphate buffer solution collocation method is：By the KH of 21mL 0.2mol/L2PO4, 78mL 0.2mol/L
Na2HPO4·12H2O mixes, and obtains the phosphate buffer solution that 0.1mol/L pH are 7.4；
Electrochemical Detection uses CHI760E electrochemical workstations (Shanghai Chen Hua instrument company).
Z300K supercentrifuges (Hermle, Germany) are used for centrifugation.
Brief description of the drawings
Fig. 1 detects the principle schematic of myostatin content.
Fig. 2 antibody/nano platinum particle solution usage (A) and probe solution and sample effect time (B) are to electrochemical signals
The influence of intensity.
Fig. 3 myostatins concentration and electrochemical signals graph of a relation.
The advantage and effect of invention
Under optimal testing conditions, the standard curve of myostatin is obtained detecting, the range of linearity and linear
Equation.When the concentration of myostatin concentration is between 0.7~25.0ng/mL, the signal intensity of system is with muscle life
The increase of inhibin concentration long and increase.The equation of linear regression for obtaining myostatin is ip=0.6536C+15.584
(ipIt is the signal intensity of system；C is the concentration of myostatin, 10-8ng/mL；N=7, R=0.999) (such as Fig. 3 institutes
Show).The method detection is limited to 0.3ng/mL (3 σ).Myostatin to concentration 3.0ng/mL carries out 7 parallel repetitions
The RSD of measure is 4.3%, shows that this law has preferable reappearance.
Specific embodiment
The present invention is further illustrated with reference to specific embodiment, but does not constitute the further limitation to inventing.
1 antibody of embodiment/influence of the nano platinum particle solution usage to electrochemical signals
Fixed probe solution and sample effect time, antibody/nano platinum particle solution usage has been investigated to Electrochemical Detection
The influence of signal.Result shows, when antibody/nano platinum particle solution usage solution usage from 20 μ of μ L to 120 L when increasing, electricity
Chemical signal strengthens with the increase of volume；When consumption is more than 90 μ L, electrochemical signals increase slows down (as shown in Figure 2 A).Cause
This, it is the optimum amount of antibody/nano platinum particle solution that 90 μ L are chosen in experiment.
The influence of the probe solution of embodiment 2 and sample effect time to electrochemical response signal
When sessile antibody/nano platinum particle solution usage is 90 μ L, probe solution and sample effect time electricity have been investigated
Influence of the chemistry to signal intensity, with the increase of action time, electrochemical signals gradually strengthen, and are 30min when action time
When, electrochemical signals maximum (as shown in Figure 2 B).Therefore, experiment chooses 30min as probe solution and sample effect time
Optimum amount.
Mice skeletal myostatin content detection after the exhausted movemeat of embodiment 3
Exhausted movemeat is carried out to mouse and is drawn materials, mice skeletal myostatin contains after detection exhausted movemeat
Amount, testing result is as shown in table 1.Result shows that the content of mice skeletal myostatin is after exhausted movemeat
4.56ng/mg, the content of control group mice skeletal muscle myostatin is 5.72ng/mg.Show, mouse after exhausted movemeat
The content of skeletal muscle myostatin decreases compared with control group.
The assay result of mice skeletal myostatin after the exhausted movemeat of table 1.
aN=7；C is control group；S exhausted movemeat groups.
Claims (2)
1. it is a kind of detect exhausted movemeat after mice skeletal myostatin content method, it is characterised in that utilize Jenner
Rice corpuscles modified electrode, recycles the effect of antibody and golden nanometer particle, prepares the gold electrode of antibody modification, obtains final product antibody/gold
Nano-particle/gold electrode；Then antibody/golden nanometer particle/gold electrode is immersed in the sample containing myostatin, then
Add antibody modification nano platinum particle；Sandwich structure is formed using the effect of myostatin and antibody, right
Nitrophenol PNP, NaBH4, ferrocenecarboxylic acid Fc system in, nano platinum particle is in NaBH4It is right that the lower catalysis PNP of effect is converted into
Amino-phenol PAP, the PAP of generation is by the Fc oxidations in solution, oxidation product 1,4-benzoquinone imines p-quinone imine and quilt
NaBH4PAP is reduced into, lasting redox is produced between such PAP and oxidation product, so that electrochemical signals are produced,
There is redox cycle in the presence of nano platinum particle, electrochemical response signal is amplified；According to the strong of electrochemical signals
The weak measure that can be realized to myostatin；Determination step is as follows：
(1) preparation of nano platinum particle
First, the glass apparatus chloroazotic acid foam washing used by nano platinum particle, dry for standby will be prepared；Then it is 1% by 7mL concentration
Platinum acid chloride solution in add 1.0mL concentration be 1% ascorbic acid, under the conditions of 80 DEG C, stir 20 minutes, when solution becomes black
Brown stops heating, obtains nano platinum particle, is cooled to room temperature, is saved backup under the conditions of 4 DEG C；
(2) preparation of antibody modification nano platinum particle
First, the 2mL nano platinum particles solution for preparing is mixed with the phosphate buffer solution of 8mL pH 7.4, the platinum for being diluted
Nano-particle, nano platinum particle 2mL and 0.5mL the myostatin antibody for then pipetting dilution mixes, under the conditions of 37 DEG C,
Concussion reaction 24h, obtains antibody/nano platinum particle compound, using supercentrifuge eccentric cleaning after, be dispersed again in PBS delay
Rush in solution, preserved at 4 DEG C；
(3) preparation of antibody modification gold electrode
First, the solution of gold nanoparticles of 10 μ L is added dropwise in the gold electrode surfaces handled well, 10h is incubated under the conditions of 4 DEG C, obtain golden
Nanoparticle Modified gold electrode；Then by the myostatin antibody drop coating of 10 μ L in golden nanometer particle modified gold electrode table
Face, under the conditions of 37 DEG C, react 24h, obtain antibody/golden nanometer particle/gold electrode, with phosphate buffer solution cleaning electrode after, at 4 DEG C
Lower preservation；
(4) detection of myostatin
By in antibody/golden nanometer particle/gold electrode sample solution of the immersion containing myostatin, reacted under the conditions of 37 DEG C
2h, then takes out electrode, is flushed three times with phosphate buffer solution, is inserted into PNP containing 5mmol/L, the 5mmol/L of pH 7.4
NaBH4In the phosphate buffer solution of 5mmol/L Fc, on electrochemical workstation, believed with cyclic voltammetry detection electrochemistry
Number, current potential is -200~600mV, uses 50mV s-1Velocity scanning；
(5) mice skeletal myostatin content detection after exhausted movemeat
Exhausted movemeat is carried out to mouse and is drawn materials, the content of mice skeletal myostatin, knot after detection exhausted movemeat
Fruit shows that the content of mice skeletal myostatin is 4.56ng/mg, control group mice Skeletal Muscle after exhausted movemeat
The content of meat amicine is 5.72ng/mg；Show, after exhausted movemeat the content of mice skeletal myostatin compared with
Control group decreases.
2. it is according to claim 1 it is a kind of detect exhausted movemeat after mice skeletal myostatin content method, its
Myostatin, myostatin antibody described in being characterised by are purchased from R＆D Systems companies；
Described chloroplatinic acid, ascorbic acid, ferrocenecarboxylic acid is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；
Described sodium borohydride NaBH4, p-nitrophenol PNP be purchased from Tianjin chemical reagent Co., Ltd；
Described PBS cushioning liquid collocation methods are：By the KH of 21mL 0.2mol/L2PO4, 78mL 0.2mol/L
Na2HPO4·12H2O mixes, and adds KCl solids so that the concentration of KCl is 0.1mol/L, and it is 7.4 to obtain 0.1mol/LpH
PBS cushioning liquid；
Described phosphate buffer solution collocation method is：By the KH of 21mL 0.2mol/L2PO4, 78mL 0.2mol/L
Na2HPO4·12H2O mixes, and obtains the phosphate buffer solution that 0.1mol/L pH are 7.4；
Described electrochemical workstation is the CHI760E electrochemical workstations of Shanghai Chen Hua instrument company；
Described supercentrifuge is Z300K supercentrifuges.
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