CN106404875B - The method for carrying out Electrochemical Detection telomerase activation using the combination of tetra- serobila of methylene blue and G- - Google Patents
The method for carrying out Electrochemical Detection telomerase activation using the combination of tetra- serobila of methylene blue and G- Download PDFInfo
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Abstract
The present invention discloses a kind of method that the combination using tetra- serobila of methylene blue and G- carries out Electrochemical Detection telomerase activation, and steps are as follows for this method: being expanded firstly, Telomerase is added in the solution containing Telomerase primer and forms tetra- serobila of G-；Then, methylene blue is added makes it in conjunction with tetra- chain body portion of G-；Finally, carrying out DPV detection to the methylene blue in solution using electrochemical workstation.The present invention is not necessarily to expensive marker DNA and carries out complicated electrode modification, can avoid at high cost, loaded down with trivial details, the defect of poor reproducibility of operation caused by labeled DNA probe and modified electrode.The present invention has many advantages, such as that at low cost, quick, easy, high sensitivity, accuracy are good.
Description
Technical field
The invention belongs to biosensor technique fields, and in particular to carry out electricity using the combination of tetra- serobila of methylene blue and G-
The method of chemical detection telomerase activation.
Background technique
Since nearly half a century, the disease incidence and the death rate of lung cancer are increasing year by year.The World Health Organization periodically announces
Data show that the morbidity and mortality of lung cancer are in the trend obviously risen in countries in the world, and developed country becomes apparent.
Countries in the world all put into the early detection and treatment of a large amount of manpower and material resources research lung cancer.Between Telomerase and human malignancies
One of the most commonly used tumor molecular marker for making it and being currently known that is closely connected, the detection of Telomerase is to cancer
Early detection, development and prognosis are of great importance.
Telomerase is the enzyme for synthesizing the telomere of end of chromosome.Telomere (telomere) is the natural of eukaryocyte chromosome
End is made of thousands of 6 base repetitive sequences (TTAGGG), is hereditary component necessary to cell.Its effect is to maintain end
Grain has enough length, guarantees the accuracy of gene information in a replication process, to prevent losing for end of chromosome hereditary information
It loses.Under normal circumstances, each cell division cycle can make telomere become shorter and shorter, just when telomere is short to a certain degree
Signal, cessation of cell division can be issued.Telomerase is made of RNA and protein subunit, is basic nucleoprotein reverse transcriptase,
In cell chromosome reproduction process, repetitive sequence can be synthesized in 3 ' end of chromosome, maintains telomere using itself RNA as template
Length.Activity inhibited of the Telomerase in normal somatic cell, but a large amount of data shows in some malignant tumours
Expression up to 85%.It since Telomerase is specifically expressed in various tumour cells, and is that most of tumour cells persistently divide
It is necessary.Therefore, telomerase activation is the good index diagnosed after most of malignant tumour, judgement more.
Traditional activity test method of telomerase mainly has telomere repeat sequence extension method, telomeric repeatamplification protocol
(TRAP), fluorescence real-time quantitative method (RT-PCR).The method that the method system establishes earliest, stability is good, and specificity is high；The disadvantage is that
Need sample size larger, sensitivity is poor, and detection time is long, is not suitable for a large amount of detections of clinical samples, in addition same position in an experiment
The dosage of element is larger, harmful to the body of testing crew.TRAP analytic approach can be realized the detection of high-throughput high sensitivity, nothing
It is suspected to be a powerful measuring method, but maintains the shortcomings that PCR amplifying technique is brought.In addition, TRAP method need using
Expensive equipment and reagent, it is relatively time consuming.Along with inhibition telomerase activation has been proposed as the potential for the treatment of human cancer
Method, and TRAP derives from the influence of product when screening effective telomerase inhibitor such as G- tetrad ligand vulnerable to PCR, because
This, there are many limitations for this method.Fluorescence real-time quantitative method (RT-PCR) measures telomerase catalytic subunit (hTERT)
MRNA has the advantages that high sensitivity, accuracy are good, but since detection means is cumbersome, needs precision instrument, higher cost, limit
Its application clinically is made.In recent years, to substitute traditional TRAP analytic approach, researchers have developed many PCR-
It is simultaneously applied in the detection of telomerase activation by free analysis method, such as optical sensor, surface plasma resonance, electroluminescent
Chemiluminescence, Electrochemical Detection and the analysis of index isothermal duplication etc., however, above-mentioned most of strategies are because Telomerase primer is consolidated
Fixedization, it is at high cost, sensitivity is low and complicated for operation the problems such as application when be more or less restricted.In addition, current is big
Most detection methods all analyze telomerase activation using cell extract, are not inconsistent more in clinical application, and can be realized original
The detection method then phoenix feathers and unicorn horns of position analysis and dynamic monitoring cell telomerase activity.The detection of telomerase activation is to cancer
Early diagnosis has important biological significance, rises to the early warning of clinically cancer with the research for treating this complicated problem in science
To facilitation.Therefore, to simple, sensitive, the inexpensive real-time monitoring in situ and inhibitor screening of telomerase activation analysis
Technology is still current urgent problem to be solved.
Summary of the invention
Goal of the invention: methylene blue and tetra- serobila of G- are utilized technical problem to be solved by the invention is to provide a kind of
In conjunction with the method for carrying out Electrochemical Detection telomerase activation.Telomerase primer, which is acted on, for Telomerase in the present invention generates G- tetra-
The combination of serobila, tetra- serobila of G- and methylene blue reduces the concentration for the methylene blue being freely present in solution, by thus drawing
The variation of electricity chemical signal measures telomerase activation, it has convenient and efficient, high sensitivity, accuracy good, unmarked etc.
Advantage.
Technical solution: in order to solve the above-mentioned technical problem, the technical scheme adopted by the invention is as follows: it is a kind of to utilize methylene
The method that blue and tetra- serobila of G- combination carries out Electrochemical Detection telomerase activation, comprising the following steps:
1) cell solution after the culture of cell and the extraction of Telomerase are cracked；
2) cell solution after cracking is added in the buffer solution containing Telomerase primer is expanded to obtain duplicate
Rich G sequence obtains the amplification solution containing tetra- serobila of G- in the presence of potassium ion；
3) methylene blue is added in the amplification solution containing tetra- serobila of G- makes it obtain solution in conjunction with tetra- serobila of G-；
4) methylene blue in solution is detected using differential pulse voltammetry (DPV).
Wherein, above-mentioned steps 1) cell be HeLa cell.
Wherein, above-mentioned steps 2) specific step is as follows: it is slow in the Telomerase that 45 μ L contain 0.1~2 μM of Telomerase primer
The HeLa cell after 5 μ L cracking is added is rushed in solution, is carried out amplification reaction at 30~37 DEG C 1~4 hour, and in potassium ion
In the presence of formed tetra- serobila of G- amplification solution.
Wherein, above-mentioned steps 2) buffer solution be contain MgCl2, KCl, Tween 20, EGTA and dNTPs pH=
8.3 20mM Tris-HCl solution, the MgCl2Initial concentration is 1.5mM, and KCl initial concentration is 63mM, at the beginning of Tween 20
Beginning concentration is 0.005% (v/v), and EGTA initial concentration is 1mM, and dNTPs initial concentration is 1~2mM.
Wherein, above-mentioned steps 2) described in Telomerase primer sequence be 5 '-AAT CCG TCG AGC AGA GTT-3 ',
This is the unique identification sequence that Telomerase is expanded.
Wherein, above-mentioned steps 3) methylene blue additional amount be 50 μ L, concentration be 1~10mM.
Wherein, above-mentioned steps 4) differential pulse voltammetry the step of be to be detected by three-electrode system, it is specific to walk
It is rapid as follows: firstly, ITO electrode is successively immersed in acetone, in ethyl alcohol and ultrapure water, and to carry out clearly within ultrasound 15~20 minutes respectively
It washes；Later, the ITO electrode after cleaning is immersed in 1mM NaOH and is modified；After 4~6 hours, the ITO of modification will be completed
Electrode, which is put into ultrapure water, to be cleaned by ultrasonic 15~20 minutes, and the ITO working electrode that surface has negative electrical charge has been obtained；Then by table
Face is put into the groove of electrolytic cell lower half portion with the ITO working electrode of negative electrical charge, will be the hole 6mm with diameter later
Electrolytic cell top half is fixed on above working electrode, and being put into O-ring seal between them prevents leakage, is 6mm in diameter
Hole in the obtained solution of step 3) is added, a platinum filament is used as to electrode, silver/silver chloride electrode is as reference electrode insertion
In solution, three-electrode system is formed with working electrode and Electrochemical Detection is carried out by DPV method.
Wherein, quantity of the above-mentioned HeLa cell in telomere enzyme buffer solution is 10~10000 cells.
The utility model has the advantages that compared with prior art, the present invention have the advantages that following characteristic and:
1) not complicated electrode modification process of the invention, it is easy to operate, it avoids and carrys out the cumbersome of band because of electrode modification
And the shortcomings that time-consuming.
(2) present invention is not necessarily to marker DNA, can avoid that DNA is marked in the prior art and causes testing cost high
Disadvantage.
(3) present invention carries out amplification extension using specific recognition of the Telomerase to its primer, and this method can be improved
Specificity.
(4) electrochemica biological sensor has many advantages, such as highly sensitive, highly compatible and low in cost, reduces experiment
Detection limit.
(5) this method can be used for the detection of actual sample, have certain clinical meaning.
Detailed description of the invention
Fig. 1 shows the method for the combination progress Electrochemical Detection telomerase activation using tetra- serobila of methylene blue and G-
Flow chart.
Fig. 2 shows telomerase activation test experience proof diagram.A: DPV curve when methylene blue is only existed；B: it is added
Telomerase primer and the DPV curve generated in conjunction with methylene blue；C: Telomerase primer is expanded and is formed with Telomerase
The DPV curve generated in conjunction with methylene blue after tetra- serobila of G-.
Fig. 3 shows the DPV curve of quantitative detection telomerase activation.Fig. 3 A: it under different amounts of HeLa cytosis, obtains
DPV curve (the number of HeLa cell: (a) 0, (b) 10, (c) 50, (d) 100, (e) 200, (f) 500, (g) 1000, (h) arrived
2000,(i)5000,(j)10000)；The canonical plotting of Fig. 3 B:DPV current peak decreasing value and number of cells；Illustration: DPV
The linear relationship of the logarithm of current peak decreasing value and number of cells.
Fig. 4 is the telomerase activation in different cells, the specific detection of Telomerase and the result of repeated experiment.Fig. 4 A
The telomere that (HeLa cell, the HeLa cell after heating, A549 cell, MCF-7 cell) extracts is shown from different cells
The activity of enzyme；Fig. 4 B show different enzymes (Telomerase, glucose oxidase, bovine serum albumin, horseradish peroxidase) with
DPV current peak decreasing value after the effect of Telomerase primer.
Specific embodiment
Below by specific embodiment and attached drawing, the present invention is further described, it is noted that for the general of this field
For logical technical staff, without departing from the principle of the present invention, several variations and modifications can also be made, these should also be regarded
To belong to the scope of protection of the present invention.
The reagent and instrument used in this experiment:
HeLa cell, other cells such as A549 cell, MCF-7 cell are bought in Shanghai Fu Xiang Biotechnology Co., Ltd,
ITO electrode, Telomerase (telomerase), methylene blue, bovine serum albumin (BSA), glucose oxidase (GOD), horseradish mistake
Oxide enzyme (HRP), electrochemical workstation (CHI660) etc. are to buy on the market.
Telomerase primer sequence: 5 '-AAT CCG TCG AGC AGA GTT-3 '
Telomerase primer is synthesized from Shanghai biotechnology Services Co., Ltd；
Embodiment 1:
The analysis method of Electrochemical Detection telomerase activation, detection step are carried out using the combination of tetra- serobila of methylene blue and G-
Suddenly it is:
1) cell culture and Telomerase extract: HeLa cell inoculation is containing 10% fetal calf serum, penicillin and streptomysin
DMEM culture medium in, and in 5%CO2, cultivate in 37 DEG C of incubators.All cells are all to be collected in exponential growth period.It will
1000000 cells are sub-packed in the EP pipe of 1.5mL, are washed twice with ice-cold phosphate buffer solution (pH=7.4), and again
It is dispersed in the ice-cold CHAPS lysis buffer of 200 μ L (10mM Tris-HCl, pH 7.5,1mM MgCl2, 1mM EGTA (second two
Bis- (the 2- amino-ethyl ether) tetraacethyls of alcohol), 0.5% (w/v) CHAPS is (in 3- [3- (gallbladder acyl aminopropyl) dimethylamino] propane sulfonic acid
Salt), in 10% (v/v) glycerol (glycerol), 0.1mM PMSF (phenylmethylsulfonyl fluoride).Cell is incubated for 30 points on ice
Then clock is centrifuged 20 minutes under 4 DEG C, the revolving speed of 12000rpm.Clear lysate is carefully transferred to after centrifugation new
In EP pipe, HeLa cell after being cracked is rapidly frozen and is stored in -80 DEG C of refrigerator；
2) the HeLa cell after cracking is added in the buffer solution containing Telomerase primer is expanded to obtain duplicate
Rich G sequence obtains the amplification solution containing tetra- serobila of G- in the presence of potassium ion: containing 2 μM of Telomerase primers in 45 μ L
The HeLa cell after cracking after 5 μ L dilution is added in telomere enzyme buffer solution, carries out amplification reaction 2 at 37 DEG C by about 100
Hour, and in the presence of potassium ion formed tetra- serobila of G- amplification solution；Wherein, the telomere enzyme buffer solution be containing
MgCl2, KCl, Tween 20, EGTA and dNTPs pH=8.3 20mM Tris-HCl solution, the MgCl2Initial concentration is
1.5mM, KCl initial concentration are 63mM, and 20 initial concentration of Tween is 0.005% (v/v), and EGTA initial concentration is 1mM,
DNTPs initial concentration is 1mM.
3) methylene blue is added in the amplification solution containing tetra- serobila of G- makes it obtain solution in conjunction with tetra- serobila of G-: containing
Methylene blue is added in the amplification solution of tetra- serobila of G- makes it in conjunction with tetra- serobila of G-: the amplification containing tetra- serobila of G- that will be obtained
Solution is mixed with the methylene blue that 50 μ L concentration are 4 μM, and methylene blue and tetra- serobila of G- is made to be combined to obtain solution.
4) modification working electrode makes its surface with negative electrical charge: firstly, ITO electrode is successively immersed in acetone, ethyl alcohol and
In ultrapure water, and ultrasound is cleaned for 15 minutes respectively.Later, cleaned ITO electrode is immersed in 1mM NaOH and is carried out
Modification.After 4 hours, the ITO electrode for completing modification is put into ultrapure water and is cleaned by ultrasonic 15 minutes, has obtained surface with negative electricity
The ITO working electrode of lotus.
5) specific steps detected using differential pulse voltammetry (DPV) to the methylene blue in solution are such as
Under: one electrolytic cell of self-control is as detecting element.Surface is put into electrolytic cell lower half portion with the ITO working electrode of negative electrical charge
Groove in, later, will be fixed on above ITO working electrode with the electrolytic cell top half that diameter is the hole 6mm, at them
Between be put into O-ring seal and prevent leakage.The solution after amplification is added in the hole that diameter is 6mm, using a platinum filament as to electricity
Pole, silver/silver chloride electrode form three-electrode system with ITO working electrode and pass through DPV method as in reference electrode insertion solution
Carry out Electrochemical Detection.Parameter: scanning voltage -0.5~0V, pulse height 0.05V, pulse width 0.05s, pulse increment is set
0.004V, pulse period 0.5s.After detection, the d curve in Fig. 3 A has been obtained.
Embodiment 2
The analysis method of Electrochemical Detection telomerase activation, detection step are carried out using the combination of tetra- serobila of methylene blue and G-
Suddenly it is:
1) cell culture and Telomerase extract: HeLa cell inoculation is containing 10% fetal calf serum, penicillin and streptomysin
DMEM culture medium in, and in 5%CO2, cultivate in 37 DEG C of incubators.All cells are all to be collected in exponential growth period.It will
1000000 cells are sub-packed in the EP pipe of 1.5mL, are washed twice with ice-cold phosphate buffer solution (pH=7.4), and again
It is dispersed in the ice-cold CHAPS lysis buffer of 200 μ L (10mM Tris-HCl, pH 7.5,1mM MgCl2, 1mM EGTA,
0.5% (w/v) CHAPS, 10% (v/v) glycerol, 0.1mM PMSF) in.Cell is incubated for 30 minutes on ice, is then existed
It 4 DEG C, is centrifuged 20 minutes under the revolving speed of 12000rpm.Clear lysate is carefully transferred in new EP pipe after centrifugation, is obtained
HeLa cell after to cracking, is rapidly frozen and is stored in -80 DEG C of refrigerator；
2) the HeLa cell after cracking is added in the buffer solution containing Telomerase primer is expanded to obtain duplicate
Rich G sequence obtains the amplification solution containing tetra- serobila of G- in the presence of potassium ion: containing 2 μM of Telomerase primers in 45 μ L
The HeLa cell after cracking after 5 μ L dilution is added in telomere enzyme buffer solution, carries out amplification reaction at 30 DEG C by about 1000
2 hours, and tetra- serobila of G- is formed in the presence of potassium ion.Wherein, the telomere enzyme buffer solution is to contain MgCl2、KCl、
The 20mM Tris-HCl solution of the pH=8.3 of Tween 20, EGTA and dNTPs, the MgCl2Initial concentration is 1.5mM, KCl
Initial concentration is 63mM, and 20 initial concentration of Tween is 0.005% (v/v), and EGTA initial concentration is 1mM, dNTPs initial concentration
For 1mM.
3) methylene blue is added in the solution after amplification makes it in conjunction with tetra- serobila of G-: by the solution after obtained amplification
The methylene blue for being 4 μM with 50 μ L concentration mixes, and is combined methylene blue with tetra- serobila of G-.
4) modification working electrode makes its surface with negative electrical charge: firstly, ITO electrode is successively immersed in acetone, ethyl alcohol and
In ultrapure water, and ultrasound is cleaned for 20 minutes respectively.Later, cleaned ITO electrode is immersed in 1mM NaOH and is carried out
Modification.After 6 hours, the ITO electrode for completing modification is put into ultrapure water and is cleaned by ultrasonic 20 minutes, has obtained surface with negative electricity
The ITO working electrode of lotus.
5) detected that specific step is as follows to the methylene blue in solution using differential pulse voltammetry (DPV): from
An electrolytic cell is made as detecting element.Surface is put into the recessed of electrolytic cell lower half portion with the ITO working electrode of negative electrical charge
In slot, later, it will be fixed on above ITO working electrode with the electrolytic cell top half that diameter is the hole 6mm, put between them
Entering O-ring seal prevents leakage.The solution after amplification is added in the hole that diameter is 6mm, using a platinum filament as to electrode,
Silver/silver chloride electrode as reference electrode insertion solution in, with ITO working electrode formed three-electrode system and by DPV method into
Row Electrochemical Detection.Parameter: scanning voltage -0.5~0V, pulse height 0.05V, pulse width 0.05s, pulse increment is set
0.004V, pulse period 0.5s.After detection, the g curve in Fig. 3 A has been obtained.
Embodiment 3
The analysis method of Electrochemical Detection telomerase activation, detection step are carried out using the combination of tetra- serobila of methylene blue and G-
Suddenly it is:
1) cell culture and Telomerase extract: HeLa cell inoculation is containing 10% fetal calf serum, penicillin and streptomysin
DMEM culture medium in, and in 5%CO2, cultivate in 37 DEG C of incubators.All cells are all to be collected in exponential growth period.It will
1000000 cells are sub-packed in the EP pipe of 1.5mL, are washed twice with ice-cold phosphate buffer solution (pH=7.4), and again
It is dispersed in the ice-cold CHAPS lysis buffer of 200 μ L (10mM Tris-HCl, pH 7.5,1mM MgCl2, 1mM EGTA,
0.5% (w/v) CHAPS, 10% (v/v) glycerol, 0.1mM PMSF) in.Cell is incubated for 30 minutes on ice, is then existed
It 4 DEG C, is centrifuged 20 minutes under the revolving speed of 12000rpm.Clear lysate is carefully transferred in new EP pipe after centrifugation, is obtained
HeLa cell after to cracking, is rapidly frozen and is stored in -80 DEG C of refrigerator；
2) the HeLa cell after cracking is added in the buffer solution containing Telomerase primer is expanded to obtain duplicate
Rich G sequence obtains the amplification solution containing tetra- serobila of G- in the presence of potassium ion: containing 2 μM of Telomerase primers in 45 μ L
The HeLa cell after cracking after 5 μ L dilution is added in telomere enzyme buffer solution, carries out amplification reaction at 37 DEG C by about 5000
2 hours, and tetra- serobila of G- is formed in the presence of potassium ion.Wherein, the telomere enzyme buffer solution is to contain MgCl2、KCl、
The 20mM Tris-HCl solution of the pH=8.3 of Tween 20, EGTA and dNTPs, the MgCl2Initial concentration is 1.5mM, KCl
Initial concentration is 63mM, and 20 initial concentration of Tween is 0.005% (v/v), and EGTA initial concentration is 1mM, dNTPs initial concentration
For 1mM.
3) methylene blue is added in the solution after amplification makes it in conjunction with tetra- serobila of G-: by the solution after obtained amplification
The methylene blue for being 4 μM with 50 μ L concentration mixes, and is combined methylene blue with tetra- serobila of G-.
4) modification working electrode makes its surface with negative electrical charge: firstly, ITO electrode is successively immersed in acetone, ethyl alcohol and
In ultrapure water, and ultrasound is cleaned for 17 minutes respectively.Later, cleaned ITO electrode is immersed in 1mM NaOH and is carried out
Modification.After 5 hours, the ITO electrode for completing modification is put into ultrapure water and is cleaned by ultrasonic 17 minutes, has obtained surface with negative electricity
The ITO working electrode of lotus.
5) specific steps detected using differential pulse voltammetry (DPV) to the methylene blue in solution are such as
Under: one electrolytic cell of self-control is as detecting element.Surface is put into electrolytic cell lower half portion with the ITO working electrode of negative electrical charge
Groove in, later, will be fixed on above ITO working electrode with the electrolytic cell top half that diameter is the hole 6mm, at them
Between be put into O-ring seal and prevent leakage.The solution after amplification is added in the hole that diameter is 6mm, using a platinum filament as to electricity
Pole, silver/silver chloride electrode form three-electrode system with ITO working electrode and pass through DPV method as in reference electrode insertion solution
Carry out Electrochemical Detection.Parameter: scanning voltage -0.5~0V, pulse height 0.05V, pulse width 0.05s, pulse increment is set
0.004V, pulse period 0.5s.After detection, the i curve in Fig. 3 A has been obtained.
DPV current peak decreasing value is defined as-Δ ip, then DPV current peak decreasing value and cell have been finally obtained
Linear relationship between several logarithms, i.e. ,-Δ ip=0.1276-0.0517log N.The range of linearity is 10~10000 cells,
Related coefficient is 0.9904.Lowest detection is calculated and is limited to 3 cells.
Embodiment 4:
The analysis method of Electrochemical Detection telomerase activation, detection step are carried out using the combination of tetra- serobila of methylene blue and G-
Suddenly it is:
1) cell culture and Telomerase extract: HeLa cell, A549 cell and MCF-7 cell be seeded in respectively containing
In the DMEM culture medium of 10% fetal calf serum, penicillin and streptomysin, and in 5%CO2, cultivate in 37 DEG C of incubators.It is all thin
Born of the same parents are to be collected in exponential growth period.1,000,000 cells are sub-packed in the EP pipe of 1.5mL, with ice-cold phosphate-buffered
Solution (pH=7.4) washes twice, and is dispersed in the ice-cold CHAPS lysis buffer of 200 μ L (10mM Tris-HCl, pH again
7.5,1mM MgCl2, 1mM EGTA (bis- (the 2- amino-ethyl ether) tetraacethyls of ethylene glycol), 0.5% (w/v) CHAPS (3- [3- (gallbladder
Acyl aminopropyl) dimethylamino] propane sulfonic acid inner salt), 10% (v/v) glycerol (glycerol), 0.1mM PMSF (benzyl sulphonyl
Fluorine) in.Cell is incubated for 30 minutes on ice, is then centrifuged 20 minutes under 4 DEG C, the revolving speed of 12000rpm.It will be clear after centrifugation
Clear lysate is carefully transferred in new EP pipe, and the cell after respectively obtaining each autothermic cracking is rapidly frozen and is stored in -80
DEG C refrigerator in；
2) cell after cracking is added in the buffer solution containing Telomerase primer is expanded to obtain duplicate richness G sequence
Column, obtain the amplification solution containing tetra- serobila of G-: in the Telomerase that 45 μ L contain 2 μM of Telomerase primers in the presence of potassium ion
The cell after cracking after being separately added into 5 μ L dilution in buffer solution, the HeLa cell after being heated and inactivated, A549 cell, MCF-7
Cell, carries out amplification reaction 2 hours at 37 DEG C by about 1000, and the amplification of tetra- serobila of G- is formed in the presence of potassium ion
Solution；Wherein, the telomere enzyme buffer solution is to contain MgCl2, KCl, Tween20, EGTA and dNTPs pH=8.3
20mM Tris-HCl solution, the MgCl2Initial concentration is 1.5mM, and KCl initial concentration is 63mM, 20 initial concentration of Tween
For 0.005% (v/v), EGTA initial concentration is 1mM, and dNTPs initial concentration is 1mM.
3) methylene blue is added in the amplification solution containing tetra- serobila of G- makes it obtain solution in conjunction with tetra- serobila of G-: containing
Methylene blue is added in the amplification solution of tetra- serobila of G- makes it in conjunction with tetra- serobila of G-: the amplification containing tetra- serobila of G- that will be obtained
Solution is mixed with the methylene blue that 50 μ L concentration are 4 μM, and methylene blue and tetra- serobila of G- is made to be combined to obtain solution.
4) modification working electrode makes its surface with negative electrical charge: firstly, ITO electrode is successively immersed in acetone, ethyl alcohol and
In ultrapure water, and ultrasound is cleaned for 15 minutes respectively.Later, cleaned ITO electrode is immersed in 1mM NaOH and is carried out
Modification.After 4 hours, the ITO electrode for completing modification is put into ultrapure water and is cleaned by ultrasonic 15 minutes, has obtained surface with negative electricity
The ITO working electrode of lotus.
5) specific steps detected using differential pulse voltammetry (DPV) to the methylene blue in solution are such as
Under: one electrolytic cell of self-control is as detecting element.Surface is put into electrolytic cell lower half portion with the ITO working electrode of negative electrical charge
Groove in, later, will be fixed on above ITO working electrode with the electrolytic cell top half that diameter is the hole 6mm, at them
Between be put into O-ring seal and prevent leakage.The solution after amplification is added in the hole that diameter is 6mm, using a platinum filament as to electricity
Pole, silver/silver chloride electrode form three-electrode system with ITO working electrode and pass through DPV method as in reference electrode insertion solution
Carry out Electrochemical Detection.Parameter: scanning voltage -0.5~0V, pulse height 0.05V, pulse width 0.05s, pulse increment is set
0.004V, pulse period 0.5s.After detection, Fig. 4 A has been obtained.
It can be seen from the figure that using A549 and HeLa cell detection go out curent change it is larger, this illustrate A549 and
Telomerase activation in HeLa cell is higher, and the telomerase activation in MCF-7 is compared with the telomerase activation in both cells
It is relatively low, and the HeLa cell heated causes enzyme to inactivate due to heating, the reduction of current signal is very faint.
Embodiment 5:
The analysis method of Electrochemical Detection telomerase activation, detection step are carried out using the combination of tetra- serobila of methylene blue and G-
Suddenly it is:
1) cell culture and Telomerase extract: HeLa cell, A549 cell and MCF-7 cell be seeded in respectively containing
In the DMEM culture medium of 10% fetal calf serum, penicillin and streptomysin, and in 5%CO2, cultivate in 37 DEG C of incubators.It is all thin
Born of the same parents are to be collected in exponential growth period.1,000,000 cells are sub-packed in the EP pipe of 1.5mL, with ice-cold phosphate-buffered
Solution (pH=7.4) washes twice, and is dispersed in the ice-cold CHAPS lysis buffer of 200 μ L (10mM Tris-HCl, pH again
7.5,1mM MgCl2, 1mM EGTA (bis- (the 2- amino-ethyl ether) tetraacethyls of ethylene glycol), 0.5% (w/v) CHAPS (3- [3- (gallbladder
Acyl aminopropyl) dimethylamino] propane sulfonic acid inner salt), 10% (v/v) glycerol (glycerol), 0.1mM PMSF (benzyl sulphonyl
Fluorine) in.Cell is incubated for 30 minutes on ice, is then centrifuged 20 minutes under 4 DEG C, the revolving speed of 12000rpm.It will be clear after centrifugation
Clear lysate is carefully transferred in new EP pipe, and the cell after respectively obtaining cracking is rapidly frozen and is stored in -80 DEG C
In refrigerator；
2) the HeLa cell after cracking is added in the buffer solution containing Telomerase primer is expanded to obtain duplicate
Rich G sequence obtains the amplification solution containing tetra- serobila of G- in the presence of potassium ion: containing 2 μM of Telomerase primers in 45 μ L
The HeLa cell after cracking after being separately added into 5 μ L dilution in telomere enzyme buffer solution, about 1000,5 μ L GOD, 5 μ L BSA,
With 5 μ L HRP, about 100 μ g are carried out amplification reaction 2 hours at 37 DEG C, and tetra- serobila of G- is formed in the presence of potassium ion
Expand solution；Wherein, the telomere enzyme buffer solution is to contain MgCl2, KCl, Tween 20, EGTA and dNTPs pH=8.3
20mM Tris-HCl solution, the MgCl2Initial concentration is 1.5mM, and KCl initial concentration is 63mM, and Tween 20 is initial dense
Degree is 0.005% (v/v), and EGTA initial concentration is 1mM, and dNTPs initial concentration is 1mM.
3) methylene blue is added in the amplification solution containing tetra- serobila of G- makes it obtain solution in conjunction with tetra- serobila of G-: containing
Methylene blue is added in the amplification solution of tetra- serobila of G- makes it in conjunction with tetra- serobila of G-: the amplification containing tetra- serobila of G- that will be obtained
Solution is mixed with the methylene blue that 50 μ L concentration are 4 μM, and methylene blue and tetra- serobila of G- is made to be combined to obtain solution.
4) modification working electrode makes its surface with negative electrical charge: firstly, ITO electrode is successively immersed in acetone, ethyl alcohol and
In ultrapure water, and ultrasound is cleaned for 15 minutes respectively.Later, cleaned ITO electrode is immersed in 1mM NaOH and is carried out
Modification.After 4 hours, the ITO electrode for completing modification is put into ultrapure water and is cleaned by ultrasonic 15 minutes, has obtained surface with negative electricity
The ITO working electrode of lotus.
5) specific steps detected using differential pulse voltammetry (DPV) to the methylene blue in solution are such as
Under: one electrolytic cell of self-control is as detecting element.Surface is put into electrolytic cell lower half portion with the ITO working electrode of negative electrical charge
Groove in, later, will be fixed on above ITO working electrode with the electrolytic cell top half that diameter is the hole 6mm, at them
Between be put into O-ring seal and prevent leakage.The solution after amplification is added in the hole that diameter is 6mm, using a platinum filament as to electricity
Pole, silver/silver chloride electrode form three-electrode system with ITO working electrode and pass through DPV method as in reference electrode insertion solution
Carry out Electrochemical Detection.Parameter: scanning voltage -0.5~0V, pulse height 0.05V, pulse width 0.05s, pulse increment is set
0.004V, pulse period 0.5s.After detection, Fig. 4 B has been obtained.
From as can be seen that electric current just has apparent reduction after only joined HeLa cell, and grape being added in Fig. 4 B
The sample of carbohydrate oxidase, bovine serum albumin and horseradish peroxidase leads to electric current due to the presence without Telomerase
Reduction it is very faint.Thus illustrate, which has preferable specificity.
Compared with current existing detection method, the detection limit of this method has a clear superiority.For example, Wuhan Polytechnic University is raw
Using the scheme of limited extension method detection telomerase activation, detection, which limits, reaches 250 cells for object and pharmaceutical engineering institute；Hebei is big
Chemistry is learned to be based on hybridizing the amplification of chain reaction signal and magnetic separation technique progress fluorescence detection telomere enzyme activity with environmental science institute
Property method, detection be limited to 1 × 105A cell；University Of Hebei's chemistry is based on constant temperature exponential amplification with environmental science institute and reacts
The method of high sensitivity detection telomerase activation, detection are limited to 50 cells.
It above are only the preferred embodiment of the invention, be not restricted to the present invention.Those skilled in the art is come
It says, other various forms of variations or variation can also be made on the basis of the above description.There is no need and unable to all
Embodiment illustrate.And the obvious changes or variations that thus scheme is extended out are still in protection of the invention
Within the scope of.
Claims (1)
1. a kind of method that the combination using tetra- serobila of methylene blue and G- carries out Electrochemical Detection telomerase activation, feature exist
In, comprising the following steps:
1) cell solution after the culture of cell and the extraction of Telomerase are cracked；The cell is HeLa cell；It is described
Quantity of the HeLa cell in telomere enzyme buffer solution is 10 ~ 10000 cells；
2) cell solution after cracking is added in the buffer solution containing Telomerase primer is expanded to obtain duplicate richness G sequence
Column, obtain the amplification solution containing tetra- serobila of G- in the presence of potassium ion；Contain 0.1 ~ 2 μM of Telomerase primer in 45 μ L
Telomere enzyme buffer solution in the Telomerase extracted in 5 μ L cells is added, it is small to carry out amplification reaction 1 ~ 4 at 30 ~ 37 DEG C
When, and in the presence of potassium ion formed tetra- serobila of G- amplification solution；The buffer solution is to contain MgCl2、KCl、Tween
20,20 mM Tris-HCl solution of pH=8.3 of EGTA and dNTPs, the MgCl2Initial concentration is 1.5 mM, and KCl is initial
Concentration is 63 mM, and 20 initial concentration of Tween is 0.005% (v/v), and EGTA initial concentration is 1 mM, and dNTPs initial concentration is 1
~ 2 mM；
3) methylene blue is added in the amplification solution containing tetra- serobila of G- makes it obtain solution in conjunction with tetra- serobila of G-；The Asia
The additional amount of methyl blue is 50 μ L, and concentration is 1 ~ 10 mM；
4) methylene blue in solution is detected using differential pulse voltammetry, the step of differential pulse voltammetry is
It is detected by three-electrode system, the specific steps are as follows: firstly, ITO electrode is successively immersed in acetone, ethyl alcohol and ultrapure water
In, and ultrasound is cleaned for 15 ~ 20 minutes respectively；Later, the ITO electrode after cleaning is immersed in 1 mM NaOH and is repaired
Decorations；After 4 ~ 6 hours, the ITO electrode for completing modification is put into ultrapure water and is cleaned by ultrasonic 15 ~ 20 minutes, surface has been obtained and has had
The ITO working electrode of negative electrical charge；Then surface is put into the groove of electrolytic cell lower half portion with the ITO working electrode of negative electrical charge
It is interior, later, it will be fixed on above working electrode with the electrolytic cell top half that diameter is 6 holes mm, be put into O between them
Type sealing ring prevents leakage, and the obtained solution of step 3) is added in the hole that diameter is 6 mm, using a platinum filament as to electrode,
Silver/silver chloride electrode forms three-electrode system with working electrode and carries out electricity by DPV method as in reference electrode insertion solution
Chemical detection.
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| CN104263725A (en) * | 2014-09-19 | 2015-01-07 | 上海市计量测试技术研究院 | Spire tetrahedral DNA nano-structural probe and telomerase electrochemical detection |
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