CN107389949A - A kind of electrochemical immunosensor preparation method for PCSK9 Protein Detections - Google Patents
A kind of electrochemical immunosensor preparation method for PCSK9 Protein Detections Download PDFInfo
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Abstract
It is the preparation method and application of the electrochemical immunosensor of the types (PCSK9) of proprotein convertases subtilisin Kexin 9 detection the present invention relates to the biomarker available for prediction and diagnosis of cardiovascular disease, belongs to technical field of electrochemical detection.It is characterized in that：Pass through nitrogen-doped graphene nanobelt (N GNRs), amination fullerene palladium nano platinum particle (n C first60/ PdPt) composite, golden ring color staphylococcal protein A (SPA), LBL self-assembly is used for PCSK9 antibody (Ab1) fixation；Then the poly- methylenum careuleum of nano platinum particle (Pt PMB) that synthesis obtains is mixed with PCSK9 antibody (Ab2), nanometer beacon is made, so as to be prepared for the electrochemical immunosensor of PCSK9 Protein Detections, the sensor is successfully used in the detection of PCSK9 albumen.The advantage of the invention is that high sensitivity, high specificity, detection is rapid, convenient.The present invention can provide new method for the detection of PCSK9 albumen, and useful information is provided for dlinial prediction and diagnosis of cardiovascular disease.
Description
Technical field：
It is proprotein convertases withered grass bacteriolyze the present invention relates to the biomarker available for prediction and diagnosis of cardiovascular disease
The preparation method and application of the electrochemical immunosensor of the plain types of Kexin 9 (PCSK9) detection, based on nitrogen-doped graphene nanometer
Band (N-GNRs), amination fullerene-palladium nano platinum particle (n-C60/ PdPt) it is used as electrode modified material, nano platinum particle-poly-
The interlayer type immunosensor that methylenum careuleum (Pt-PMB) is prepared as nanometer beacon, for detecting PCSK9, belongs to Electrochemical Detection
Field.
Background technology：
At present, angiocardiopathy because its incidence of disease, disability rate and fatal rate it is higher, threaten the health of the mankind, have a strong impact on
People's quality of life, have become the main reason for causing human death.The types of proprotein convertases subtilisin Kexin 9
(PCSK9) it is a kind of secreting type serine protease mainly synthesized by liver, belongs to proprotein convertases family.With low-density
Lipoprotein receptor (LDLR) combines, and reduces surface of hepatocytes LDLR, and then make liver cell under LDL-C particle Scavenging activities
Drop, plasma LDL-C levels rise, causes the occurrence and development of atherosclerosis, promotes the generation of cardiocerebrovasculaevents events.Grind
Study carefully and show, the PCSK9 in human serum be can be used for one of cardiovascular disease diagnosis and prediction its onset risk it is reliable and good
Biochemical marker.Therefore, PCSK9 in human serum is quantitatively detected to be of great significance cardiovascular disease.
Although the traditional detection method for PCSK9 has the methods of enzyme linked immunosorbent assay (ELISA), fluorescent immune method,
But these methods have many deficiencies, such as sample handling processes are excessively cumbersome, analysis time is longer, sensitivity is limited.In recent years
Come, electrochemical immunosensor technology has quick, high sensitivity, simple to operate, stability is good as emerging detection technique
The features such as, and it is widely used to the fields such as biochemical analysis, environmental monitoring, clinical research and food quality detection, this side
Detection of the method to PCSK9 provides new Research Thinking.
In electrochemical immunosensor analytical technology, in order to improve the sensitivity of detection and shorten the response time, meet
The clinically quantitative quick detection of trace materials, at present more using the sensor response patterns of " sandwich " type.Its principle is base
In structure load primary antibody (Ab1)-measured target Ab- marking nano beacons (Ab2) sandwich immunoassay compound analysis method.How
It is easy, quick to realize that it is by the major criterion of its popularization and application that detection is carried out to target substance.To realize this purpose, electrode
The selection of decorative material and nanometer beacon is particularly important.In recent years, in carbon nanomaterial, N-GNRs is as new carbon
Material, there is good electric conductivity and excellent physical and chemical performance, the extensive use in electrochemical biosensor technology.It is based on
Above advantage, this experiment increase electric conductivity using N-GNRs as electrode modified material.In order to increase the sensitivity of sensor, this
Experiment introduces palladium nano platinum particle (PdPt), because it has good electric conductivity, larger specific surface area, good biology
The features such as compatibility, electrochemical immunosensor electric signal can be amplified, while in order to increase PdPt supported quantity, employ ratio
The big n-C of surface area60N-C is formed with reference to PdPt60/ PdPt nano composite materials carry out immobilized more albumen.In addition, antibody exists
Fixation on electrode is to build the important step of immunosensor, therefore this problem uses staphylococcal protein A (SPA), and it is
The cell-wall components of staphylococcus aureus, there are four avtive spots, it has specific binding immunoglobulin G (IgG)
Nonantigenic Fc part and antigen binding site is dissociated.Therefore, antibody can be oriented by SPA and is incorporated in electrode table
On face, and the problem of overcome the random orientation and steric hindrance of antibody, and greatly reduce the time of capture antibody.Meanwhile this
Problem intends being used as nanometer beacon using the redox probe Pt-PMB that synthesizes first, because it has uniform form, by force
Oxidation-reduction quality and good electric conductivity, traditional electropolymerization methylenum careuleum form heterogeneity can be overcome, easily taken off with biomolecule
From the shortcomings that.And because there is PtNPs presence, the stability of nanometer beacon is added, metal-ammonia can also be passed through with antibody
Polymerization of olefin using catalyst is combined, and the structure of interlayer type immunosensor is implemented in combination with by antigen and antibody specific.
The project, which establishes a simple, quick detection method, realizes special, super sensitivity detection to PCSK9.For
Clinical cardiovascular disease prediction and diagnosis provide corresponding reference.
The content of the invention：
1. it is an object of the invention to provide a kind of preparation method of the electrochemical immunosensor for PCSK9 albumen with answering
With its feature comprises the following steps：
(1) amination fullerene-palladium nano platinum particle (n-C60/ PdPt) composite preparation；
(2) nano platinum particle-poly- methylenum careuleum (Pt-PMB)-Ab2The preparation of nanometer beacon；
(3) immunosensor is established, determines PCSK9 albumen, draws standard curve.
2. n-C of the present invention60The preparation process of/PdPt nano composite materials specifically includes following steps：
First, by 2mg amination fullerenes (n-C60) be dissolved in 2mL 0.1M hydrochloric acid (HCl), at room temperature ultrasound 2
Hour, obtain uniform suspension.Then solution is heated with stirring to 100 DEG C, is separately added into 1mL 4mM chlorine palladium acid sodium
(Na2PdCl4), 1mL 4mM Platinous Potassium Chlorides (K2PtCl4) and 150mg ascorbic acid (AA), and continuously stirred at 100 DEG C
2.5 hour.After naturally cooling to room temperature, obtained product is centrifuged, with milli-Q water 3 times.Final sediment is disperseed
Further used in 1mL ultra-pure waters.
3. Pt-PMB-Ab of the present invention2The preparation process of nanometer beacon specifically includes following steps：
(1) preparation of Pt-PMB nano composite materials：
First, at room temperature by 2mL 4.9mM methylenum careuleum (MB), 3mL 2.28mM DTABs (DTAB)
With 600 μ L 0.1M HCl micella is formed after mixing is stirred vigorously 10 minutes.Then by the chloroplatinic acid (H of 160 μ L 5%2PtCl6·
6H2O) rapidly join in micella, and be continuously stirred at room temperature 6 hours.Finally obtained compound is centrifuged and washed 3 times
Afterwards, it is dispersed in 2mL ultra-pure waters and is used for next step.
(2)Pt-PMB-Ab2The preparation of nanometer beacon：
By prepared 1mL Pt-PMB and 100 μ L 1mgmL-1PCSK9 antibody mixes, and is gently mixed 12 at 4 DEG C
Hour.Then, add 1% bovine serum albumin(BSA) (BSA) and block nonspecific binding site.Finally, by the Pt-PMB- of gained
Ab2 bioconjugates, with phosphate buffer PBS (0.1M, pH 7.4) centrifuge washing 3 times, it is dispersed in 1mL PBS (0.1M, pH
7.4) stored in solution and in 4 DEG C.
4. of the present invention establish electrochemical immunosensor, PCSK9 albumen is determined, draws standard curve, its feature
It is to comprise the following steps：
(1) respectively with 0.3 and 0.05 μm of Al2O3Powder by polishing electrode into minute surface, then respectively by ultra-pure water, anhydrous
Order each 5min of ultrasound electrode of ethanol, ultra-pure water, drying at room temperature are standby；
(2) by 10 μ L 0.4mg mL-1The graphene nanobelt (N-GNRs) of electrode modified material N doping is added dropwise in electrode
Surface, drying at room temperature.
(3) by 8 μ L n-C60Dry electrode surface, drying at room temperature are arrived in the modification of-PdPt nano composite materials.
(4) 6 μ L 0.1mg mL are added dropwise in the electrode modified-1Golden ring color staphylococcal protein A (SPA), incubated at 37 DEG C
Educate 40min.
(5) electrode washing after incubation is totally added dropwise to 8 μ L PCSK9 antibody afterwards with ultra-pure water, 60min is incubated at 37 DEG C.
(6) electrode washing after incubation is totally added dropwise to 6 μ L, 1% BSA solution incubation at room temperature 30min afterwards with ultra-pure water.
(7) electrode cleaning buffer solution (the 10mM Na after above-mentioned BSA is closed2HPO4, 2mM KH2PO4, 37mM
NaCl, 2.7mM KCl, pH 7.4) rinse well and dried in nitrogen.
(8) the target PCSK9 of various concentrations is added dropwise and 37 ° of incubation 60min is placed on electrode.
(9) 8 μ L Pt-PMB-Ab are added dropwise on electrode after the drying2Nanometer beacon is placed in 37 DEG C of incubation 60min.
(10) it is placed in nitrogen and dries after the electrode after incubation is rinsed well with cleaning buffer solution.
(11) electrode is placed in 5mL, 0.1M PBS (0.1M Na2HPO4, 0.1M KH2PO4, 0.1M KCl) in carry out table
Sign, measure differential pulse voltammetric current value.
(12) it is linear according to gained current variation value and PCSK9 protein concentrations, drawing curve.
Compared with prior art, the preparation method of a kind of electrochemical immunosensor for PCSK9 albumen of the invention
With application, its protrude the characteristics of be：
(1) it is incorporated into N-GNRs as electrode modified material in the preparation of electrochemical immunosensor, improves sensing
The electric conductivity of device, accelerate electron transmission, and then improve sensitivity and the biocompatibility of electrochemical immunosensor, introduce n-
C60/ PdPt increases electric conductivity, specific surface area and the biocompatibility of sensor, while PdPt can be coordinated by metal-amino
With reference to immobilized more albumen, using the SPA with antibody orientating function come sessile antibody, increase immune response efficiency, reduction
The time of antibody is captured, further improves the sensitivity of sensor；
(2) the redox probe Pt-PMB synthesized first has uniform form, strong oxidation also as nanometer beacon
Originality and good electric conductivity, can be produced, amplified signal；
(3) electrochemical immunosensor prepared by this method can provide effectively letter for the prevention and detection of clinical cardiovascular disease
Breath, contributes to the diagnosis and prevention of cardiovascular disease.
(4) electrochemical immunosensor prepared by this method is due to using the specific binding between antibody antigen, having
Good specificity, its preparation process is simple, detecting step is less, and detection speed is very fast, is easy to implement commercialization, is advantageous to push away
Enter the development of translational medicine.
Brief description of the drawings：
Fig. 1 is the structure schematic diagram of the electrochemical immunosensor of the present invention.
Fig. 2 be the present invention electrode modified material and signal material different synthesis steps field emission scanning electron microscope figure,
Transmission electron microscope picture, EDS figures and XPS figures.
Fig. 3 is the differential pulse voltammetry and its peak that the electrochemical immunosensor of the present invention obtains when detecting PCSK9
The linear relationship of current value and log concentration.
Embodiment：
The present invention is further elaborated with reference to specific embodiment, it should be appreciated that these embodiments are merely to illustrate
The present invention rather than limitation the scope of the present invention.
Embodiment 1
Step 1. by the multi-walled carbon nanotube (N-MWCNT) of 50mg N dopings be dispersed in 5mL volume ratios be 9: 1 sulfuric acid-
Nitric acid (H2SO4-H3PO4) in mixture, then reacted 10 minutes at 140 DEG C.Then add 25mg potassium permanganate (KMnO4).It
Mixture is handled 10 minutes at 65 DEG C afterwards.Finally centrifuge, be dried in vacuo after washing at 35 DEG C；
Step 2. is respectively with 0.3 and 0.05 μm of Al2O3Polishing electrode into minute surface, is then pressed ultra-pure water, nothing by powder respectively
Order each 5min of ultrasound electrode of water-ethanol, ultra-pure water, drying at room temperature are standby.
Step 3. is by 10 μ L 0.4mg mL-1The graphene nanobelt (N-GNRs) of electrode modified material N doping is added dropwise
Electrode surface, drying at room temperature；
Step 4. is by 8 μ L n-C60Dry electrode surface, drying at room temperature are arrived in the modification of-PdPt nano composite materials；
6 μ L 0.1mg mL are added dropwise in the electrode modified by step 5.-1Golden ring color staphylococcal protein A (SPA), 37
DEG C be incubated 40min；
Step 6. electrode washing after incubation is totally added dropwise afterwards with ultra-pure water 8 μ L PCSK9 antibody, in 37 DEG C of incubations
60min；
Step 7. electrode washing after incubation is totally added dropwise afterwards with ultra-pure water 6 μ L, 1% BSA solution incubation at room temperature
30min；
Step 8. by above-mentioned BSA close after electrode cleaning buffer solution (10mM Na2HPO4, 2mM KH2PO4, 37mM
NaCl, 2.7mM KCl, pH 7.4) rinse well and dried in nitrogen；
The target PCSK9 of various concentrations is added dropwise step 9. is placed in 37 ° of incubation 60min on electrode；
8 μ L Pt-PMB-Ab are added dropwise on the electrode of step 10. after the drying2Nanometer beacon is placed in 37 DEG C of incubation 60min；
Step 11. is placed in nitrogen after the electrode after incubation is rinsed well with cleaning buffer solution and dried；
Electrode is placed in 5mL, 0.1M PBS (0.1M Na by step 12.2HPO4, 0.1M KH2PO4, 0.1M KCl) in carry out
Characterize, measure differential pulse voltammetric current value；
Step 13. is linear according to gained current variation value and PCSK9 protein concentrations, drawing curve；Measure
As a result show PCSK9 concentration in 100fg mL-1To 100ng mL-1In the range of it is linear, linearly dependent coefficient is
0.9998, detection is limited to 0.033pg mL-1。
Step 14. is by the sensor of the present invention in 4 DEG C of preservations, discontinuity detection sensor current response, after storing 28 days
Current-responsive is still the 88.6% of initial current, and surface probe has good stability；
Step 15. present invention takes immunosensor 5 prepared by same batch, under the same conditions to 100pgmL-1's
PCSK9 albumen is measured respectively, each determination of electrode 3 times, and as a result the relative standard deviation of response current is less than 4.07%,
Illustrate that difference is small in sensor batch, sensor reappearance is good；
The present invention is used to detect the interfering material in PCSK9 and blood plasma by step 16., the results showed that the interference in blood plasma
Electric current analog value of the current-responsive value of material well below PCSK9, illustrates the specific good of sensor, strong antijamming capability,
The interference of other materials can be excluded.
Described above is only the preferred embodiment of the present invention, it is noted that for the common skill of the art
For art personnel, under the precondition for not departing from the principle of the invention, some improvements and modifications can also be made, these improve and
Retouching also should be regarded as protection scope of the present invention.
Claims (4)
1. a kind of electrochemical immunosensor preparation method for PCSK9 Protein Detections, it is characterised in that comprise the following steps：
(1) amination fullerene-palladium nano platinum particle (n-C60/ PdPt) composite preparation；
(2) nano platinum particle-poly- methylenum careuleum (Pt-PMB)-Ab2The preparation of nanometer beacon；
(3) immunosensor is established, determines PCSK9 albumen, draws standard curve.
2. n-C according to claim 160The preparation process of/PdPt nano composite materials specifically includes following steps：
First, by 2mg amination fullerenes (n-C60) be dissolved in 2mL 0.1M hydrochloric acid (HCl), ultrasound 2 is small at room temperature
When, obtain uniform suspension.Then solution is heated with stirring to 100 DEG C, is separately added into 1mL 4mM chlorine palladium acid sodium
(Na2PdCl4), 1mL 4mM Platinous Potassium Chlorides (K2PtCl4) and 150mg ascorbic acid (AA), and continuously stirred at 100 DEG C
2.5 hour.After naturally cooling to room temperature, obtained product is centrifuged, with milli-Q water 3 times.Final sediment is disperseed
Further used in 1mL ultra-pure waters.
3. Pt-PMB-Ab according to claim 12The preparation process of nanometer beacon specifically includes following steps：
(1) preparation of Pt-PMB nano composite materials：
First, at room temperature by 2mL 4.9mM methylenum careuleum (MB), 3mL 2.28mM DTABs (DTAB) and
600 μ L0.1M HCl form micella after mixing is stirred vigorously 10 minutes.Then by the chloroplatinic acid (H of 160 μ L 5%2PtCl6·
6H2O) rapidly join in micella, and be continuously stirred at room temperature 6 hours.Finally obtained compound is centrifuged and washed 3 times
Afterwards, it is dispersed in 2mL ultra-pure waters and is used for next step.
(2)Pt-PMB-Ab2The preparation of nanometer beacon：
By prepared 1mL Pt-PMB and 100 μ L 1mg mL-1PCSK9 antibody mixes, and it is small at 4 DEG C to be gently mixed 12
When.Then, add 1% bovine serum albumin(BSA) (BSA) and block nonspecific binding site.Finally, by the Pt-PMB-Ab of gained2
Bioconjugates, with phosphate buffer PBS (0.1M, pH7.4) centrifuge washing 3 times, it is molten to be dispersed in 1mL PBS (0.1M, pH7.4)
Stored in liquid and in 4 DEG C.
4. according to claim 1 establish electrochemical immunosensor, PCSK9 albumen is determined, draws standard curve, it is special
Sign is to comprise the following steps：
(1) respectively with 0.3 and 0.05 μm of Al2O3Powder by polishing electrode into minute surface, then respectively by ultra-pure water, absolute ethyl alcohol,
Order each 5min of ultrasound electrode of ultra-pure water, drying at room temperature are standby；
(2) by 10 μ L 0.4mg mL-1The graphene nanobelt (N-GNRs) of electrode modified material N doping is added dropwise in electrode table
Face, drying at room temperature.
(3) by 8 μ L n-C60Dry electrode surface, drying at room temperature are arrived in the modification of-PdPt nano composite materials.
(4) 6 μ L 0.1mg mL are added dropwise in the electrode modified-1Golden ring color staphylococcal protein A (SPA), 37 DEG C incubation
40min。
(5) electrode washing after incubation is totally added dropwise to 8 μ L PCSK9 antibody afterwards with ultra-pure water, 60min is incubated at 37 DEG C.
(6) electrode washing after incubation is totally added dropwise to 6 μ L, 1% BSA solution incubation at room temperature 30min afterwards with ultra-pure water.
(7) electrode cleaning buffer solution (the 10mM Na after above-mentioned BSA is closed2HPO4, 2mM KH2PO4, 37mM NaCl,
2.7mM KCl, pH7.4) rinse well and dried in nitrogen.
(8) the target PCSK9 of various concentrations is added dropwise and 37 ° of incubation 60min is placed on electrode.
(9) 8 μ L Pt-PMB-Ab are added dropwise on electrode after the drying2Nanometer beacon is placed in 37 DEG C of incubation 60min.
(10) it is placed in nitrogen and dries after the electrode after incubation is rinsed well with cleaning buffer solution.
(11) electrode is placed in 5mL, 0.1M PBS (0.1M Na2HPO4, 0.1M KH2PO4, 0.1M KCl) in characterized, survey
Measure differential pulse voltammetric current value.
(12) it is linear according to gained current variation value and PCSK9 protein concentrations, drawing curve.
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| CN108896632B (en) * | 2018-04-11 | 2021-01-05 | 东南大学 | Photoelectrochemical immunosensor for determining alpha fetoprotein content and preparation method and application thereof |
| CN109187510A (en) * | 2018-09-05 | 2019-01-11 | 浙江理工大学 | A method of ancient times woolen knitwear is detected based on Electrochemiluminescince |
| CN109187510B (en) * | 2018-09-05 | 2020-10-16 | 浙江理工大学 | Method for detecting ancient wool fabric based on electrochemical luminescence method |
| CN109187511B (en) * | 2018-09-10 | 2021-04-20 | 浙江理工大学 | Method for detecting ancient silk fabric based on electrochemical luminescence method |
| CN109187511A (en) * | 2018-09-10 | 2019-01-11 | 浙江理工大学 | A method of Ancient Silk Textile is detected based on Electrochemiluminescince |
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| CN109540983A (en) * | 2019-01-22 | 2019-03-29 | 重庆医科大学 | It is a kind of for detecting the novel electrochemical Biosensors of 2,6 sialylated glycan of α |
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