CN106226374A - A kind of detection method of ErbB-2 concentration - Google Patents
A kind of detection method of ErbB-2 concentration Download PDFInfo
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- CN106226374A CN106226374A CN201610684899.2A CN201610684899A CN106226374A CN 106226374 A CN106226374 A CN 106226374A CN 201610684899 A CN201610684899 A CN 201610684899A CN 106226374 A CN106226374 A CN 106226374A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses the detection method of a kind of ErbB-2 concentration, comprise the following steps: gold electrode is polished, clean, be dried；Gold electrode inserts self assembly in polypeptide solution, and sulfydryl hexanol is closed；Take variable concentrations HER 2 to drop to above-mentioned gold electrode surfaces respectively and react, then react with aptamers；Molybdate dropping is reacted the gold electrode to be measured of root much in gold electrode surfaces；Respectively gold electrode is measured with square wave voltammetry, obtains a plurality of square wave volt-ampere curve, the peak current in each curve is done standard curve with corresponding HER 2 concentration；Take HER 2 to be measured and drop to be assembled with the gold electrode surfaces reaction of polypeptide, then react with aptamers, molybdate is dripped in gold electrode surfaces, measures to obtain square wave volt-ampere curve with square wave voltammetry, by peak current and standard curve control, record the concentration of HER 2.This detection method is convenient and swift, selectivity good, highly sensitive, detection range width.
Description
Technical field
The present invention relates to biosensor technique field, particularly relate to the inspection of a kind of human epidermal growth factor acceptor-2 concentration
Survey method.
Background technology
Human epidermal growth factor acceptor-2 (human epidermal growth factor receptor-2；HER-
2) it is a kind of receptor TYR kinases, multiple human cancer suffers from the expression of height.It is the distinctive mark of breast carcinoma
Protein, closely related with the transitivity of breast carcinoma, can effectively judge the rear situation of breast carcinoma.
The receptor TYR kinases of epidermal growth factor associated protein family in several cancers as main treatment mesh
It is marked on during epithelium increases and plays an important role.EebB (HER) family member of overexpression and sudden change can cause substantial amounts of pernicious
Tumor, the method for traditional mensuration HER-2 has surface plasma body resonant vibration, SABC, euzymelinked immunosorbent assay (ELISA) etc..These detection sides
Although method is the sensitiveest effectively, but technology is required height, take time and effort, and instrument is had certain requirement, each
There is certain limitation.
Summary of the invention
The technical problem to be solved is, overcomes the deficiency and defect mentioned in background above technology, it is provided that one
Plant the detection side of human epidermal growth factor acceptor-2 concentration convenient and swift, selectivity good, highly sensitive, detection range is wide
Method.
For solving above-mentioned technical problem, the technical scheme that the present invention proposes is:
The detection method of a kind of human epidermal growth factor acceptor-2 concentration, comprises the following steps:
(1) carry out gold electrode polishing, clean, obtain the gold electrode through pretreatment after drying；
(2) gold electrode through pretreatment is inserted and can carry out specific recognition with human epidermal growth factor acceptor-2
Carrying out self assembly in polypeptide solution, self assembly is rinsed after completing, and then closes with sulfydryl hexanol, is again rinsed,
To the gold electrode being assembled with polypeptide；
(3) take the human epidermal growth factor acceptor-2 of variable concentrations to drop to many step (2) gained respectively and be assembled with
The gold electrode surfaces of polypeptide is reacted, and is rinsed after having reacted, and then enters with energy and human epidermal growth factor acceptor-2
The aptamers reaction of row specific recognition, has reacted backlash and has washed unreacted aptamers off, obtained the gold electrode after many modifications；
(4) molybdate solution is dripped the gold electrode surfaces after step (3) gained is modified and carry out reaction generation phosphomolybdic acid
Salt precipitates, and obtains many gold electrodes to be measured；
(5) respectively step (4) institute root gold electrode to be measured much is measured with square wave voltammetry, obtains a plurality of square wave volt
Peace curve, then does standard by the peak current in each square wave volt-ampere curve with corresponding human epidermal growth factor acceptor-2 concentration
Curve；
(6) take the human epidermal growth factor acceptor to be measured-2 of unknown concentration to drop to step (2) gained and be assembled with polypeptide
Gold electrode surfaces react, be rinsed after having reacted, then with can and human epidermal growth factor acceptor-2 carry out spy
The opposite sex identify aptamers reaction, reacted backlash and washed unreacted aptamers off, obtain unknown concentration human epidermal growth because of
Gold electrode after the modification of sub-receptor-2；
(7) molybdate solution is dripped the gold electrode surfaces after unknown concentration human epidermal growth factor acceptor-2 is modified
Carry out reaction and generate phosphomolybdate precipitation, obtain unknown concentration human epidermal growth factor acceptor-2 gold electrode to be measured；
(8) with square wave voltammetry to step (7) gained unknown concentration human epidermal growth factor acceptor-2 gold electrode to be measured
It is measured, obtains square wave volt-ampere curve, then by the peak current in this square wave volt-ampere curve and step (5) gained standard curve
Compare, i.e. record the concentration of the human epidermal growth factor acceptor-2 to be measured of step (6) described unknown concentration.
The detection method of the present invention is by " gold electrode surfaces pretreatment "-" self-assembling polypeptide "-" closing of sulfydryl hexanol "-" suitable
Ligand modified " multiple steps such as-" phosphomolybdate precipitation "-" sweeping square wave volt-ampere curve " combine and detect HER-2 concentration,
Easy to operate, selectivity good, highly sensitive, and detection range is wider.The present invention uses and can carry out specific recognition with HER-2
Polypeptide carry out self assembly in gold electrode surfaces, containing sulfydryl in polypeptide, the S in sulfydryl is closed by Au-S bond with gold electrode,
Obtain being assembled with the gold electrode of polypeptide；With sulfydryl hexanol, gold electrode surfaces is closed, prevent from occurring non-in gold electrode surfaces
Specific adsorption；Then react with the aptamers that HER-2 carries out specific recognition at gold electrode surfaces dropping energy, this adaptation
Body is a kind of single stranded DNA, has high-affinity and specificity, and the specific binding target molecule HER-2 of energy, containing big in aptamers
The phosphate group of amount, these phosphate groups can react generation phosphomolybdate precipitation with molybdate, produce electrochemical signals, HER-2
Concentration be directly proportional (such as Fig. 2) to the peak current intensity in the electrochemical signals of generation；Aptamers in traditional detection method
Generally only as target recognition probe molecule, and aptamers has not only acted as the work of target recognition probe molecule in the present invention
With, also as signal probe molecule, enormously simplify detecting step.By variable concentrations HER-2 gold electrode is swept square wave volt-ampere
Curve obtains standard curve after being analyzed processing, and this standard curve is that the concentration of straight line (such as Fig. 3), HER-2 is the biggest then
The corresponding peak current in square wave volt-ampere curve is the biggest；HER-2 gold electrode to unknown concentration sweeps square wave volt-ampere curve again, will
Peak current in gained square wave volt-ampere curve and standard curve control, record the concentration of unknown concentration HER-2.The present invention utilizes energy
The polypeptide carrying out specific recognition with HER-2 replaces in traditional detection method to resist, and utilization can carry out specificity knowledge with HER-2
Other aptamers replaces two in traditional detection method to resist, and simplifies detecting step, reduces testing cost, improves detection choosing
Selecting property and sensitivity.
Above-mentioned detection method, it is preferred that in described step (2), described can enter with human epidermal growth factor acceptor-2
The peptide sequence of row specific recognition is CKLRLEWNR.
Above-mentioned detection method, it is preferred that in described step (4) and step (7), described molybdate is sodium molybdate or molybdic acid
Potassium, the concentration of described molybdate is 1-5mmol/L, and the temperature of described reaction is 32-42 DEG C, and the response time is 0.5-1h.
Above-mentioned detection method, it is preferred that in described step (3) and step (6), described aptamers sequence is
GCAGCGGTGTGGGG。
Above-mentioned detection method, it is preferred that in described step (5) and step (8), the concrete mensuration of described square wave voltammetry
Condition is: with the sulfuric acid solution of 0.5mol/L as electrolyte, in 0-0.5V voltage range, be measured with the frequency of 15Hz.
Above-mentioned detection method, it is preferred that in described step (1), described carries out gold electrode polishing, cleans, is dried
Detailed process is: first by the polishing powder polishing 4-6min that gold electrode particle diameter is 0.3 μm, then with the polishing that particle diameter is 0.05 μm
Powder carries out second polishing, is cleaned by the secondary water of the gold electrode after second polishing, then with the ultrasonic 1-3min of dehydrated alcohol,
Again be rinsed, then with the ultrasonic 1-3min of secondary water, ultrasonic after take out gold electrode, then dry up with nitrogen.
Above-mentioned detection method, it is preferred that in described step (2), described is inserted into energy by the gold electrode through pretreatment
With the detailed process carrying out self assembly in the polypeptide solution that human epidermal growth factor acceptor-2 carries out specific recognition it is: will be through
The gold electrode inversion insertion crossing pretreatment can carry out specific recognition with human epidermal growth factor acceptor-2 equipped with 8-12 μ g/mL
Polypeptide solution centrifuge tube in, under the conditions of 32-42 DEG C react 8-24h carry out self assembly.
Above-mentioned detection method, it is preferred that in described step (2), what described sulfydryl hexanol was closed specifically comprises the following steps that
Gold electrode after self assembly is inverted in the centrifuge tube inserted equipped with 0.8-1.2mmol/L sulfydryl hexanol, 32-42 DEG C of condition
Lower closing 0.4-0.6h.
Above-mentioned detection method, it is preferred that in described step (3), described in take the human epidermal growth factor of variable concentrations
Receptor-2 drops to be assembled with the gold electrode surfaces of polypeptide respectively to carry out reaction and specifically refers to: taking concentration is 0ng/mL, 0.01ng/
The human epidermal growth factor acceptor-2 of mL, 0.05ng/mL, 0.1ng/mL and 5ng/mL drops to multiple be assembled with polypeptide respectively
Gold electrode surfaces, react 0.8-1.2h.
Above-mentioned detection method, it is preferred that in described step (3), described and energy and human epidermal growth factor acceptor-2
The detailed process of the aptamers reaction carrying out specific recognition is: can carry out specificity with human epidermal growth factor acceptor-2
The aptamers identified, with rotating speed centrifugal treating 1-3min of 10000-14000rpm, is then used secondary water dissolution, is obtained aptamers
Solution, then gained aptamers solution is added drop-wise to gold electrode surfaces, react 0.8-1.2h.
Compared with prior art, it is an advantage of the current invention that:
(1) present invention is selected and can carry out the polypeptide of specific recognition with HER-2 and be combined in the table of gold electrode by self assembly
Face, owing to containing sulfydryl in peptide molecule, this sulfydryl can carry out self assembly with gold electrode by Au-S key, polypeptide is assembled into gold
Electrode surface, this polypeptide can capture target molecule HER-2 albumen with specificity, eliminate modification loaded down with trivial details in existing detection method
One contragradience is rapid, and the method selectivity is good.
(2) this method can survey the HER-2 that concentration is 0.01ng/mL-5ng/mL, and HER-2 dense in normal human cells
Degree is 2-15ng/mL, and in patient with breast cancer, HER-2 is at concentrations up to 15-75ng/mL, and the method for the present invention is to after dilution
Blood serum of patients with human breast carcinoma has enough detection sensitivities.
(3) present invention uses and can carry out the aptamers of specific recognition to the gold electrode after HER-2 modifies with HER-2
Again modifying, replace two in traditional detection method to resist by this aptamers, this aptamers is the most not only made
For the target recognition probe molecule of HER-2, and as electrochemical signals probe molecule so that the operation of this detection method is simpler
Single, cost is lower.
(4) the detection method detection range width of the present invention, it is not necessary to by precision instrument and equipment, and it is suitable to may extend to other
Part sensor, for detecting the concentration of different albumen or little molecule.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
In having technology to describe, the required accompanying drawing used is briefly described, it should be apparent that, the accompanying drawing in describing below is the present invention
Some embodiments, for those of ordinary skill in the art, on the premise of not paying creative work, it is also possible to according to
These accompanying drawings obtain other accompanying drawing.
Fig. 1 is the principle schematic of the detection method of the present invention.
Fig. 2 is the square wave volt-ampere curve of the gold electrode in embodiment 1 after variable concentrations HER-2 modification.
Fig. 3 is canonical plotting corresponding to gold electrode after variable concentrations HER-2 modifies in embodiment 1.
Detailed description of the invention
For the ease of understanding the present invention, below in conjunction with Figure of description and preferred embodiment, the present invention is made more complete
Face, describe meticulously, but protection scope of the present invention is not limited to embodiment in detail below.
Unless otherwise defined, the implication that all technical term used hereinafter is generally understood that with those skilled in the art
Identical.Technical term used herein is intended merely to describe the purpose of specific embodiment, is not intended to limit the present invention
Protection domain.
Unless otherwise specified, the various raw materials used in the present invention, reagent, instrument and equipment etc. all can pass through city
Field is commercially available or can be prepared by existing method.
Embodiment 1
A kind of embodiment of the detection method of human epidermal growth factor acceptor-2 concentration of the present invention, its principle schematic is such as
Shown in Fig. 1.This detection method specifically includes following steps:
(1) gold electrode pretreatment: first by the polishing powder polishing 5min that many gold electrode particle diameters are 0.3 μm, then use grain
Footpath is that the polishing powder of 0.05 μm carries out second polishing, and the gold electrode after polishing is first clear with secondary water (water after twice distillation)
Wash, then with the ultrasonic 2min of dehydrated alcohol, then with the ultrasonic 2min of secondary water, confirm that gold electrode surfaces is without taking out after remaining polishing powder
Gold electrode secondary water is rinsed, and then dries up with nitrogen, and root is through the gold electrode of pretreatment much.
(2) self-assembling polypeptide: being inverted by the many gold electrodes through pretreatment and insert equipped with 100 μ L, concentration is 10 μ g/mL
The polypeptide solution that sequence is CKLRLEWNR centrifuge tube in, it is ensured that every gold electrode is all inserted perpendicularly in solution and and solution
Being fully contacted, react 12h and carry out self assembly after fixing under the conditions of 37 DEG C, self assembly completes backlash and washes gold electrode surfaces off not
Polypeptide in assembling, is then inverted the gold electrode after self assembly in the centrifuge tube inserted equipped with 1mmol/L sulfydryl hexanol,
Close 0.5h under the conditions of 37 DEG C, wash away the sulfydryl hexanol that gold electrode surfaces is unnecessary after having closed, obtain many and be assembled with polypeptide
Gold electrode.
(3) gold electrode modify: take concentration be 0ng/mL, 0.01ng/mL, 0.05ng/mL, 0.1ng/mL, 0.5ng/mL,
The HER-2 of 1ng/mL and 5ng/mL drops to the many gold electrode surfaces being assembled with polypeptide respectively to carry out reacting 1h, after having reacted
It is rinsed, then by aptamers that sequence is GCAGCGGTGTGGGG with rotating speed centrifugal treating 2min of 12000rpm, with two
Secondary water dissolution, obtains aptamers solution, then gained aptamers solution is added drop-wise to gold electrode surfaces, reacts 1h, has reacted backlash
Wash unreacted aptamers off, obtain the gold electrode after many modifications.
(4) phosphomolybdate precipitation is generated: take the sodium molybdate solution that 5 μ L concentration are 1mmol/L and drip respectively at above-mentioned many
Gold electrode surfaces after modification, reacts 30min at 42 DEG C, generate sodium phosphomolybdate precipitation, obtain many gold electrodes to be measured.
(5) standard curve is done: with the sulfuric acid solution of 0.5mol/L as electrolyte, in the range of 0-0.5V, with the frequency of 15Hz
Many gold electrodes to be measured are measured respectively by rate with square wave voltammetry, obtain a plurality of square wave volt-ampere curve, then with each square wave
Peak current in volt-ampere curve is vertical coordinate, does standard curve with corresponding HER-2 concentration for abscissa.Wherein, variable concentrations
The square wave volt-ampere curve of the gold electrode after HER-2 modification is as shown in Figure 2；Gold electrode after variable concentrations HER-2 modifies is corresponding
As it is shown on figure 3, as seen from Figure 3, this standard curve is straight line to canonical plotting, the concentration of HER-2 and corresponding square wave volt-ampere
Peak point current in curve is directly proportional.
(6) HER-2 gold electrode to be measured is modified: takes the HER-2 to be measured of unknown concentration and drops to step (2) gained and be assembled with many
The gold electrode surfaces of peptide carries out reacting 1h, is rinsed after having reacted, and is then the aptamers of GCAGCGGTGTGGGG by sequence
With rotating speed centrifugal treating 2min of 12000rpm, use secondary water dissolution, obtain aptamers solution, then gained aptamers solution is dripped
It is added to gold electrode surfaces, reacts 1h, reacted backlash and washed unreacted aptamers off, after obtaining the modification of unknown concentration HER-2
Gold electrode.
(7) HER-2 gold electrode to be measured generates phosphomolybdate precipitation: drip sodium molybdate solution in above-mentioned unknown concentration HER-
Gold electrode surfaces after 2 modifications carries out reaction and generates sodium phosphomolybdate precipitation, obtains unknown concentration HER-2 gold electrode to be measured.
(8) HER-2 concentration is recorded: with the sulfuric acid solution of 0.5mol/L as electrolyte, in the range of 0-0.5V, with 15Hz's
Frequency, is measured with square wave voltammetry gold electrode to be measured to above-mentioned unknown concentration HER-2, obtains square wave volt-ampere curve, then
Peak current in this square wave volt-ampere curve is compareed with step (5) gained standard curve, i.e. records the to be measured of unknown concentration
The concentration of HER-2, the concentration of this HER-2 to be measured is the abscissa concentration value that the peak point current recorded in standard curve is corresponding.
Embodiment 2
A kind of embodiment of the detection method of human epidermal growth factor acceptor-2 concentration of the present invention, its principle schematic is such as
Shown in Fig. 1.This detection method specifically includes following steps:
(1) gold electrode pretreatment: first by the polishing powder polishing 6min that many gold electrode particle diameters are 0.3 μm, then use grain
Footpath is that the polishing powder of 0.05 μm carries out second polishing, and the gold electrode after polishing is first clear with secondary water (water after twice distillation)
Wash, then with the ultrasonic 3min of dehydrated alcohol, then with the ultrasonic 3min of secondary water, confirm that gold electrode surfaces is without taking out after remaining polishing powder
Gold electrode secondary water is rinsed, and then dries up with nitrogen, and root is through the gold electrode of pretreatment much.
(2) self-assembling polypeptide: being inverted by the many gold electrodes through pretreatment and insert equipped with 100 μ L, concentration is 12 μ g/mL
The polypeptide solution that sequence is CKLRLEWNR centrifuge tube in, it is ensured that every gold electrode is all inserted perpendicularly in solution and and solution
Being fully contacted, react 8h and carry out self assembly after fixing under the conditions of 42 DEG C, self assembly completes backlash and washes gold electrode surfaces off not
Polypeptide in assembling, is then inverted the gold electrode after self assembly and inserts the centrifuge tube equipped with 1.2mmol/L sulfydryl hexanol
In, close 0.6h under the conditions of 42 DEG C, wash away the sulfydryl hexanol that gold electrode surfaces is unnecessary after having closed, obtain many and be assembled with polypeptide
Gold electrode.
(3) gold electrode modify: take concentration be 0ng/mL, 0.01ng/mL, 0.05ng/mL, 0.1ng/mL, 0.5ng/mL,
The HER-2 of 1ng/mL and 5ng/mL drops to the many gold electrode surfaces being assembled with polypeptide respectively to carry out reacting 1.2h, has reacted
After be rinsed, then by aptamers that sequence is GCAGCGGTGTGGGG with rotating speed centrifugal treating 3min of 14000rpm, use
Secondary water dissolution, obtains aptamers solution, then gained aptamers solution is added drop-wise to gold electrode surfaces, reacts 1.2h, has reacted
Unreacted aptamers is washed in backlash off, obtains the gold electrode after many modifications.
(4) phosphomolybdate precipitation is generated: take the potassium molybdate solution that 5 μ L concentration are 5mmol/L and drip after above-mentioned modification
Gold electrode surfaces, reacts 60min at 42 DEG C, generate phosphomolybdic acid potassium precipitation, obtain many gold electrodes to be measured.
(5) standard curve is done: with the sulfuric acid solution of 0.5mol/L as electrolyte, in the range of 0-0.5V, with the frequency of 15Hz
Many gold electrodes to be measured are measured respectively by rate with square wave voltammetry, obtain a plurality of square wave volt-ampere curve, then with each square wave
Peak current in volt-ampere curve is vertical coordinate, does standard curve with corresponding HER-2 concentration for abscissa.
(6) HER-2 gold electrode to be measured is modified: takes the HER-2 to be measured of unknown concentration and drops to step (2) gained and be assembled with many
The gold electrode surfaces of peptide carries out reacting 1.2h, is rinsed after having reacted, and is then the adaptation of GCAGCGGTGTGGGG by sequence
Body, with rotating speed centrifugal treating 3min of 14000rpm, uses secondary water dissolution, obtains aptamers solution, then by gained aptamers solution
It is added drop-wise to gold electrode surfaces, reacts 1.2h, reacted backlash and washed unreacted aptamers off, obtain unknown concentration HER-2 and modify
After gold electrode.
(7) HER-2 gold electrode to be measured generates phosphomolybdate precipitation: drip potassium molybdate solution in above-mentioned unknown concentration HER-
Gold electrode surfaces after 2 modifications carries out reaction and generates phosphomolybdic acid potassium precipitation, obtains unknown concentration HER-2 gold electrode to be measured.
(8) HER-2 concentration is recorded: with the sulfuric acid solution of 0.5mol/L as electrolyte, in the range of 0-0.5V, with 15Hz's
Frequency, is measured with square wave voltammetry gold electrode to be measured to above-mentioned unknown concentration HER-2, obtains square wave volt-ampere curve, then
Peak current in this square wave volt-ampere curve is compareed with step (5) gained standard curve, i.e. records the to be measured of unknown concentration
The concentration of HER-2, the concentration of this HER-2 to be measured is the abscissa concentration value that the peak point current recorded in standard curve is corresponding.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.All within the spirit and principles in the present invention, that is made any repaiies
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (10)
1. a detection method for human epidermal growth factor acceptor-2 concentration, comprises the following steps:
(1) carry out gold electrode polishing, clean, obtain the gold electrode through pretreatment after drying；
(2) gold electrode through pretreatment is inserted and can carry out the polypeptide of specific recognition with human epidermal growth factor acceptor-2
Carrying out self assembly in solution, self assembly is rinsed after completing, and then closes with sulfydryl hexanol, is again rinsed, obtains group
Gold electrode equipped with polypeptide；
(3) take the human epidermal growth factor acceptor-2 of variable concentrations to drop to many step (2) gained respectively and be assembled with polypeptide
Gold electrode surfaces react, be rinsed after having reacted, then with can and human epidermal growth factor acceptor-2 carry out spy
The aptamers reaction that the opposite sex identifies, has reacted backlash and has washed unreacted aptamers off, obtained the gold electrode after many modifications；
(4) by molybdate solution dropping step (3) gained modify after gold electrode surfaces carry out reaction generate phosphomolybdate sink
Form sediment, obtain many gold electrodes to be measured；
(5) respectively step (4) institute root gold electrode to be measured much is measured with square wave voltammetry, obtains a plurality of square wave volt-ampere bent
Line, then does standard bent by the peak current in each square wave volt-ampere curve with corresponding human epidermal growth factor acceptor-2 concentration
Line；
(6) take the human epidermal growth factor acceptor to be measured-2 of unknown concentration to drop to step (2) gained and be assembled with the gold of polypeptide
Electrode surface reacts, and is rinsed after having reacted, and then carries out specificity with energy and human epidermal growth factor acceptor-2
The aptamers reaction identified, has reacted backlash and has washed unreacted aptamers off, obtained unknown concentration human epidermal growth factor and be subject to
Gold electrode after body-2 modification；
(7) gold electrode surfaces that molybdate solution drips after unknown concentration human epidermal growth factor acceptor-2 is modified is carried out
Reaction generates phosphomolybdate precipitation, obtains unknown concentration human epidermal growth factor acceptor-2 gold electrode to be measured；
(8) with square wave voltammetry, step (7) gained unknown concentration human epidermal growth factor acceptor-2 gold electrode to be measured is carried out
Measure, obtain square wave volt-ampere curve, then the peak current in this square wave volt-ampere curve is carried out with step (5) gained standard curve
Comparison, i.e. records the concentration of the human epidermal growth factor acceptor-2 to be measured of step (6) described unknown concentration.
The detection method of human epidermal growth factor acceptor-2 concentration the most according to claim 1, it is characterised in that: described
In step (2), described to carry out the peptide sequence of specific recognition with human epidermal growth factor acceptor-2 be CKLRLEWNR.
The detection method of human epidermal growth factor acceptor-2 concentration the most according to claim 1, it is characterised in that: described
In step (4) and step (7), described molybdate is sodium molybdate or potassium molybdate, and the concentration of described molybdate is 1-5mmol/L, institute
The temperature stating reaction is 32-42 DEG C, and the response time is 0.5-1h.
The detection method of human epidermal growth factor acceptor-2 concentration the most according to claim 1, it is characterised in that: described
In step (3) and step (6), described aptamers sequence is GCAGCGGTGTGGGG.
The detection method of human epidermal growth factor acceptor-2 concentration the most according to claim 1, it is characterised in that described
In step (5) and step (8), the concrete condition determination of described square wave voltammetry is: with the sulfuric acid solution of 0.5mol/L for electrolysis
Liquid, in 0-0.5V voltage range, is measured with the frequency of 15Hz.
The detection method of human epidermal growth factor acceptor-2 concentration the most according to claim 1, it is characterised in that described
The detailed process in step (1), described carry out gold electrode polishing, clean, being dried is: be first 0.3 μm by gold electrode particle diameter
Polishing powder polishing 4-6min, then carry out second polishing, by after second polishing with the polishing powder that particle diameter is 0.05 μm
Gold electrode secondary water cleans, and then with the ultrasonic 1-3min of dehydrated alcohol, is again rinsed, then with the ultrasonic 1-of secondary water
3min, ultrasonic after take out gold electrode, then dry up with nitrogen.
The detection method of human epidermal growth factor acceptor-2 concentration the most according to claim 1, it is characterised in that described
In step (2), described being inserted into by the gold electrode through pretreatment can carry out specificity with human epidermal growth factor acceptor-2
The detailed process carrying out self assembly in the polypeptide solution identified is: is inverted by the gold electrode through pretreatment and inserts equipped with 8-12 μ
In the centrifuge tube of the polypeptide solution that g/mL can carry out specific recognition with human epidermal growth factor acceptor-2,32-42 DEG C of condition
Lower reaction 8-24h carries out self assembly.
The detection method of human epidermal growth factor acceptor-2 concentration the most according to claim 1, it is characterised in that described
In step (2), described sulfydryl hexanol close specifically comprise the following steps that by after self assembly gold electrode be inverted insert equipped with
In the centrifuge tube of 0.8-1.2mmol/L sulfydryl hexanol, under the conditions of 32-42 DEG C, close 0.4-0.6h.
The detection method of human epidermal growth factor acceptor-2 concentration the most according to claim 1, it is characterised in that described
In step (3), described in take the human epidermal growth factor acceptor-2 of variable concentrations and drop to be assembled with the gold electrode of polypeptide respectively
Surface carries out reaction and specifically refers to: take the people that concentration is 0ng/mL, 0.01ng/mL, 0.05ng/mL, 0.1ng/mL and 5ng/mL
Skins growth factor receptors-2 drops to multiple gold electrode surfaces being assembled with polypeptide respectively, reacts 0.8-1.2h.
10., according to the detection method of human epidermal growth factor acceptor-2 concentration according to any one of claim 1-9, it is special
Levy and be, in described step (3), described with can and human epidermal growth factor acceptor-2 to carry out the aptamers of specific recognition anti-
The detailed process answered is: will can carry out the aptamers of specific recognition with 10000-with human epidermal growth factor acceptor-2
Rotating speed centrifugal treating 1-3min of 14000rpm, then uses secondary water dissolution, obtains aptamers solution, then by molten for gained aptamers
Drop is added to gold electrode surfaces, reacts 0.8-1.2h.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106950270A (en) * | 2017-03-16 | 2017-07-14 | 中南大学 | A kind of CEA Concentration Testings probe and preparation method and application, and CEA Concentration Testing biology sensors |
| CN107064258A (en) * | 2017-02-20 | 2017-08-18 | 中南大学 | The method that electric signal and its electrochemical aptamer sensor measure HER2 of self assembly amplified signal are produced based on DNA |
| CN109738417A (en) * | 2019-01-25 | 2019-05-10 | 中南大学 | A method of tumour cell is detected using porous gold nanosphere |
| CN109824773A (en) * | 2019-02-07 | 2019-05-31 | 段振学 | 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of people is mutated and application |
| WO2019184247A1 (en) * | 2018-03-27 | 2019-10-03 | 苏州长光华医生物医学工程有限公司 | Electrochemiluminescence kit for detecting mechano-growth factor and its e peptide and preparation method therefor |
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| CN101036055A (en) * | 2004-10-06 | 2007-09-12 | 威尔斯达特生物制剂公司 | Detection of elevated levels of Her-2/neu protein on circulating cancer cells and treatment |
| 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 |
| CN105784796A (en) * | 2016-03-03 | 2016-07-20 | 青岛大学 | Sensitive lysozyme measuring method of aptasensor based on gold/molybdenum disulfide/graphene nanocomposite |
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| CN101036055A (en) * | 2004-10-06 | 2007-09-12 | 威尔斯达特生物制剂公司 | Detection of elevated levels of Her-2/neu protein on circulating cancer cells and treatment |
| 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 |
| CN105784796A (en) * | 2016-03-03 | 2016-07-20 | 青岛大学 | Sensitive lysozyme measuring method of aptasensor based on gold/molybdenum disulfide/graphene nanocomposite |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107064258A (en) * | 2017-02-20 | 2017-08-18 | 中南大学 | The method that electric signal and its electrochemical aptamer sensor measure HER2 of self assembly amplified signal are produced based on DNA |
| CN107064258B (en) * | 2017-02-20 | 2019-06-28 | 中南大学 | The method of the electrochemical aptamer sensor measurement HER2 of electric signal and its self assembly amplified signal is generated based on DNA |
| CN106950270A (en) * | 2017-03-16 | 2017-07-14 | 中南大学 | A kind of CEA Concentration Testings probe and preparation method and application, and CEA Concentration Testing biology sensors |
| WO2019184247A1 (en) * | 2018-03-27 | 2019-10-03 | 苏州长光华医生物医学工程有限公司 | Electrochemiluminescence kit for detecting mechano-growth factor and its e peptide and preparation method therefor |
| CN109738417A (en) * | 2019-01-25 | 2019-05-10 | 中南大学 | A method of tumour cell is detected using porous gold nanosphere |
| CN109824773A (en) * | 2019-02-07 | 2019-05-31 | 段振学 | 8 albuminoid 3 of epidermal growth factor receptor kinase substrate of people is mutated and application |
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| CN106226374B (en) | 2018-10-16 |
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