CN106483281A - Renewable electrochemical immunosensor preparation method for sCD40L detection - Google Patents
Renewable electrochemical immunosensor preparation method for sCD40L detection Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The present invention relates to can be used to predicting and diagnosing acute coronary syndrome the preparation method of renewable electrochemical immunosensor that detects of biomarker i.e. soluble CD 40 ligand (sCD40L), belong to technical field of electrochemical detection.It is characterized in that：Multi-walled carbon nano-tubes polyethyleneimine golden nanometer particle nano-complex (c MWCNTs PEI AuNPs) initially with carboxyl-functional is used for immobilized sCD40L antibody as base material, the capture to sCD40L is realized, and then quantitative determination is carried out to sCD40L.As c MWCNTs PEI AuNPs nano-complex is prepared simply, electric conductivity is good, with preferable stability and larger specific surface area, therefore can immobilized substantial amounts of antibody securely, and using antibody and the specific recognition of antigen, make the electrochemical immunosensor of structure have stronger specificity.It is high that the present invention has sensitivity, high specificity, convenient and swift and can be repeated several times the advantage for using, and provides new method for the detection of sCD40L, is that dlinial prediction and diagnosing acute coronary syndrome provide useful information.
Description
Technical field：
The present invention relates to a kind of for can be good as prediction and diagnosing acute coronary syndrome and reliable biochemical
Mark is the preparation method of the electrochemical immunosensor that soluble CD 40 ligand (sCD40L) is detected, based on carboxyl-functional
Multi-walled carbon nano-tubes (c-MWCNTs), polyethyleneimine (PEI) and golden nanometer particle (AuNPs) nano composite material direct
The reproducible immunosensor of type, for the detection of sCD40L, belongs to field of electrochemical detection.
Background technology：
At present, angiocardiopathy because its incidence of disease, disability rate and fatal rate higher, threaten the mankind health, have become as
Cause the main cause of human death, wherein acute coronary syndrome is clinically common, serious angiocardiopathy.
SCD40L is a member in tumor necrosis factor superfamily, belongs to I type transmembrane glycoprotein, can promote the amplification of cell and move
Move.There are some researches show, the sCD40L in human serum can be used for its morbidity wind of acute coronary syndrome early diagnosis and prediction
Danger a reliability and good biochemical marker.Therefore, the sCD40L in quantitative determination human serum is to prediction and diagnosing acute
Coronary syndrome is of great significance.
The traditional detection method of protein has immunocytochemical method (ICC), Immunohistochemical Method (IHC), Western Immuno
The methods such as blot hybridization (WB), but there are many deficiencies in these methods, such as sample handling processes are loaded down with trivial details, analysis time is longer,
Instrument or expensive reagents, sensitivity are more low, are not suitable for conventional clinical detection.In recent years, electrochemical immunosensor
Because receiving much concern the features such as which is convenient and swift, sensitive, and it is widely used to biochemical analysis, environmental monitoring, clinical research
With fields such as food quality detections.
In the application of electrochemical immunosensor, realize detecting target substance to reach simply and quickly
Purpose, suitable electrode modified material should be selected.In recent years, multi-walled carbon nano-tubes CNT (MWCNTs) is excellent because of which
The features such as electric conductivity, stronger adsorption capacity, good electrochemical stability and larger specific surface area, it is widely used in electrification
Learn in immunosensor.But the presence due to its π-pi-electron, forms the interaction of Van der Waals force, with stronger hydrophobicity,
Which is caused to disperse uneven, easy reunion in many solvents, so which is received perhaps in the application of electrochemical immunosensor
Many restrictions.In order to the dispersiveness of MWCNTs is improved, reduce its agglomeration, on the one hand carboxyl is carried from c-MWCNTs because of which,
Increased its dispersiveness；On the other hand using branched PEI (containing a lot of amino in branch birds of the same feather flock together compound), with good
Water solubility, combine with c-MWCNTs, not only further increase MWCNTs dispersiveness in a solvent, and it is provided
A large amount of amino lay a good foundation for the modification further of material.Golden nanometer particle (AuNPs) is with good electric conductivity, larger
Specific surface area, stronger adsorption capacity, good biocompatibility the features such as, with amplifying electrochemical immunosensor telecommunications
Number function, the sensitivity of electrochemical immunosensor can be improved further, obtained in electrochemical immunosensor extensively
Application.The present invention not only makes electric signal amplify further using the property of above-mentioned material, and is resisted by AuNPs and sCD40L
Body forms Au-NH2Key, so as to fixing sCD40L antibody, and can keep the activity of this antibody, so as to realize determining sCD40L
Amount detection.As base material c-MWCNTs-PEI-AuNPs nano composite material electrochemical properties are stable, which is anti-with sCD40L
Body is firmly combined with, and in alkaline solution, antibody antigen dissociates, and antibody is unaffected with the connection of electrode surface, makes electrochemistry
Immunosensor has reproducibility and can repeatedly use.
The present invention is base material based on c-MWCNTs-PEI-AuNPs nano-complex, sets up a kind of for biological sample
The preparation method of the renewable electrochemical immunosensor of the Direct-type of middle sCD40L detection, is the sCD40L in Organism Samples
Easily and fast quantitative determination provides a kind of new method, is that the clinically prediction of acute coronary artery syndrome and diagnosis provide reference.
Content of the invention：
It is an object of the invention to provide a kind of renewable electrochemistry of the sCD40L quantitative determination in biological sample is exempted from
The preparation method of epidemic disease sensor, its feature are comprised the following steps：
(1) carboxyl function multi-walled carbon nano-tube (c-MWCNTs)-polyethyleneimine (PEI)-golden nanometer particle (AuNPs)
The preparation of base material；
(2) renewable electro-chemistry immunity biology sensor is set up, sCD40L is determined, draw calibration curve.
The preparation process of c-MWCNTs-PEI-AuNPs nano-complex of the present invention, its feature are comprised the following steps：
Weigh in the ultra-pure water of 2mg c-MWCNTs to 2mL, ultrasonic 1-3h so as to be uniformly dispersed.It is being stirred continuously lower adding
Enter appropriate N-hydroxy-succinamide (NHS) and 1- ethyl -3- (3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution
Its carboxyl is activated, after stirring 30min, adds 100 μ L PEI solution to continue stirring 2-5h.After above-mentioned material washing repeatedly, point
It is dispersed in 2mL ultra-pure water, is successively separately added into 100 μ L HAuCl4·6H2O (1%) and the NaBH of 900 μ L (30mM)4Solution is stirred
Mix overnight, be dispersed in 2mL ultra-pure water after washing repeatedly, you can c-MWCNTs-PEI-AuNPs nano-complex is obtained, by which
Be dispersed in 2mL ultra-pure water be stored in standby in 4 DEG C of refrigerators.
The heretofore described sCD40L concentration that sets up renewable electrochemical immunosensor, determine in biological sample,
Draw calibration curve, it is characterised in that comprise the following steps：
(1) respectively with 0.3 μm and 0.05 μm of Al2O3Then polishing electrode to minute surface is used ultrapure in right amount by powder respectively
Electrode is cleaned by ultrasonic each 5min by said sequence by water, absolute ethyl alcohol, ultra-pure water, and drying at room temperature is standby；
(2) the c-MWCNTs-PEI-AuNPs nano-complex for preparing 6 μ L is dripped in electrode surface, drying at room temperature；
(3) the sCD40L antibody dropping of 6 μ L is placed in incubation 10h in 4 DEG C of refrigerators in the electrode surface after modification；
(4), after being washed away the unconjugated sCD40L antibody of the electrode surface after incubation with ultra-pure water, 6 μ L cow's serums are dripped
Albumin (BSA, 0.25%) solution incubated at room 30min；
(5), after being washed away BSA unnecessary for electrode surface with ultra-pure water, the sCD40L of 6 μ L variable concentrations is dripped respectively
Electrode surface is incubated 45min；
(6), after being washed away the sCD40L not combined with sCD40L antibody with ultra-pure water, electrode is placed in the iron cyaniding of 5mM
Potassium solution (5mM K3[Fe(CN)6]、5mM K4[Fe(CN)6], 0.1M KCl) in characterized, use differential pulse voltammetry
(DPV) its current-responsive value is measured；
(7) linear with the logarithm of sCD40L concentration according to gained peak current difference, drawing curve；
(8) electrode for detecting sCD40L is put in dissociation solution (30mM NaOH) after rinse 60s and takes out, use ultra-pure water
Careful flushing, is placed in 4 DEG C of refrigerators and stores for future use.
Compared with prior art, the present invention is a kind of renewable electrochemistry for sCD40L quantitative determination in biological sample
The preparation method of immunosensor, its project feature be：
(1) c-MWCNTs-PEI-AuNPs nano composite material is incorporated into the system of electrochemical immunosensor as substrate
In standby, electric conductivity is not only increased, accelerate electron transmission, and the supported quantity of biomolecule is increased, and then improve electricity
The sensitivity of chemo-immunity sensor.
(2) renewable electrochemical immunosensor prepared by this method is due to its base material (c-MWCNTs-PEI-
AuNPs preparation process) is simple, convenient and electrochemical properties are stable, and antibody is firmly combined with base material.In alkalescence condition
Under, antibody is dissociated with antigen, and antibody is not affected substantially with the combination of base material, therefore has excellent stability, weight
Existing property and reproducibility, can be repeated several times use.
(3) renewable electrochemical immunosensor prepared by this method can be for clinic to the pre- of acute coronary syndrome
Anti- and diagnosis provides effective information, contributes to diagnosis and the prevention of acute coronary syndrome.
(4) renewable electrochemical immunosensor prepared by this method is due to using the specificity knot between antibody antigen
Close, with good specificity, its preparation process is simple, detecting step is less, detection speed is very fast, facilitates implementation commercialization,
So as to advance the development of translational medicine.
Description of the drawings：
Fig. 1 is the structure schematic diagram of renewable electrochemical immunosensor in the present invention.
Fig. 2 is the transmission electron microscope figure of different synthesis steps of base material in the present invention, energy spectrum diagram and ultraviolet-can
See abosrption spectrogram.
Fig. 3 is that the DPV that the renewable electrochemical immunosensor of the present invention is obtained when soluble CD 40 ligand is detected is bent
Line and its linear relationship of peak current difference and log concentration.
Specific 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 restriction the scope of the present invention.
Embodiment 1
Step 1. is weighed in the ultra-pure water of 2mg c-MWCNTs to 2mL, ultrasonic 1-3h so as to be uniformly dispersed.Constantly stirring
Mix the appropriate EDC and NHS solution of lower addition and its carboxyl is activated, after magnetic agitation 30min, add 100 μ L PEI solution to continue to stir
Mix 2-5h.After above-mentioned material washing repeatedly, it is dispersed in 2mL ultra-pure water, is successively separately added into 100 μ L HAuCl4·6H2O
(1%) NaBH of and 900 μ L4(30mM) it is stirred overnight, is dispersed in 2mL ultra-pure water after washing repeatedly, you can obtain c-
MWCNTs-PEI-AuNPs nano-complex, is dispersed in being stored in 2mL ultra-pure water standby in 4 DEG C of refrigerators；
Step 2. is respectively with 0.3 μm and 0.05 μm of Al2O3Then polishing electrode to minute surface is surpassed by powder respectively with appropriate
Electrode is respectively cleaned by ultrasonic 5min in the above sequence for pure water, absolute ethyl alcohol, ultra-pure water, and drying at room temperature is standby；
Step 3. takes the c-MWCNTs-PEI-AuNPs nano-complex of the above-mentioned 6 μ L for preparing and drips in electrode surface,
Drying at room temperature；
Step 4. drips the sCD40L antibody of 6 μ L after the electrode surface after modification, is placed in incubation 10h in 4 DEG C of refrigerators；
After the unconjugated sCD40L antibody of the electrode surface after incubation is washed away by step 5. with ultra-pure water, 6 μ L are dripped
BSA (0.25%) solution incubated at room 30min；
After BSA unnecessary for electrode surface after incubation is washed away by step 6. with ultra-pure water, 6 μ L are dripped on electrode respectively not
37 DEG C of incubation 45min are placed in the sCD40L of concentration；
After the sCD40L not combined with sCD40L antibody is washed away by step 7. with ultra-pure water, the potassium ferricyanide is placed on molten
Liquid (5mM K3[Fe(CN)6]、5mM K4[Fe(CN)6], 0.1M KCl) in characterized, measure its current-responsive value with DPV；
, drawing linear with the logarithm of sCD40L concentration that step 8. tries to achieve its difference according to gained peak point current
Curve；Measurement result shows sCD40L concentration in 10fg mL-1-100pg mL-1In the range of linear, linearly dependent coefficient
Square (R2) it is 0.99, detection is limited to 3fg mL-1(S/N=3)；
As a result step 9. shows the interference in blood plasma by the present invention for detecting sCD40L and the interfering material in blood plasma
Electric current analog value of the current-responsive value of material well below sCD40L, illustrates that the specificity of sensor is good, strong antijamming capability,
The interference of other materials can be excluded；
During step 10. is placed in the sensor in the present invention in refrigerator, 4 DEG C preserve, and discontinuity detection sensor current is rung
Should, after storing 28 days, current-responsive is still the 89.53% of initial current, shows that sensor has good stability；
5 electrochemical immunosensors of the present invention are used for detecting same concentration (1pg mL by step 11.-1)
SCD40L, its relative standard deviation are 1.38%, show that this sensor has good reappearance；
The present invention is used for detecting same concentration (1pg mL by step 12.-1) sCD40L after, be put into dissociation solution (30mM
NaOH take out after rinse 60s in), repeat the above steps after carefully being rinsed with ultra-pure water, as a result show which is repeated 5 times current value
Still 95.79% for initial current, shows that this sensor is good as the stable, antibody of material and the strong bonded of material have
Reproducibility, can be recycled for multiple times.
Claims (3)
1. the renewable electrochemical immunosensor preparation method that one kind is detected for soluble CD 40 ligand (sCD40L), its are special
Levy and be to comprise the following steps：
(1) carboxyl function multi-walled carbon nano-tube (c-MWCNTs)-polyethyleneimine (PEI)-golden nanometer particle (AuNPs) substrate
The preparation of material；
(2) renewable electrochemical immunosensor is set up, sCD40L is determined, draw calibration curve.
2. the preparation process of c-MWCNTs-PEI-AuNPs nano-complex according to claim 1, it is characterised in that include
Following steps：
In the ultra-pure water of c-MWCNTs to the 2mL for weighing 2mg, ultrasonic 1-3h so as to be uniformly dispersed.It is being stirred continuously lower addition N-
HOSu NHS (NHS) and 1- ethyl -3- (3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution activate its carboxylic
Base, adds 100 μ L PEI solution to continue stirring 2-5h after stirring 30min.After above-mentioned material washing repeatedly, it is dispersed in
In 2mL ultra-pure water, 100 μ L HAuCl are successively separately added into4·6H2O (1%) and the NaBH of 900 μ L (30mM)4Solution is stirred
At night, it is dispersed in 2mL ultra-pure water after washing repeatedly, you can obtain c-MWCNTs-PEI-AuNPs nano-complex, disperseed
It is stored in 2mL ultra-pure water standby in 4 DEG C of refrigerators.
3. according to claim 1 set up renewable electrochemical immunosensor, determine sCD40L, draw calibration curve,
It is characterized in that comprising the following steps：
(1) respectively with 0.3 μm and 0.05 μm of Al2O3Powder by polishing electrode to minute surface, then respectively with appropriate ultra-pure water, no
Water-ethanol, ultra-pure water, by said sequence, electrode are respectively cleaned by ultrasonic 5min, and drying at room temperature is standby；
(2) the c-MWCNTs-PEI-AuNPs nano-complex for preparing 6 μ L is dripped in electrode surface, drying at room temperature；
(3) the sCD40L antibody of 6 μ L is dripped after the electrode surface after modification, is placed in incubation 10h in 4 DEG C of refrigerators；
(4), after being washed away the unconjugated sCD40L antibody of the electrode surface after incubation with ultra-pure water, 6 μ L bovine serum albumins are dripped
(BSA, 0.25%) solution incubated at room 30min in vain；
(5), after being washed away BSA unnecessary for the electrode surface after incubation with ultra-pure water, the sCD40L of 6 μ L variable concentrations is dripped respectively
It is added in electrode surface incubation 45min；
(6), after being washed away the sCD40L not combined with sCD40L antibody with ultra-pure water, the potassium ferricyanide solution (5mM of 5mM is placed in
K3[Fe(CN)6]、5mM K4[Fe(CN)6], 0.1M KCl) in characterized, with differential pulse voltammetry (DPV) measure its electricity
Stream response；
(7) linear with the logarithm of sCD40L concentration according to gained peak current difference, drawing curve；
(8) electrode for detecting sCD40L is put in dissociation solution (30mM NaOH) after rinse 60s and takes out, careful with ultra-pure water
Rinse, be placed in 4 DEG C of refrigerators and store for future use.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107543924A (en) * | 2017-09-06 | 2018-01-05 | 重庆医科大学 | Biology sensor detection myeloperoxidase based on copper palladium platinum mesh nanometer material |
| CN107621492A (en) * | 2017-09-06 | 2018-01-23 | 重庆医科大学 | Biology sensor preparation method for the detection of the sialylated glycans of α 2,3 |
| CN108398477A (en) * | 2018-01-22 | 2018-08-14 | 昆明理工大学 | A kind of active electrochemical method of measurement phosphoric acid enol pyruvic acid carboxylase |
| CN109342526A (en) * | 2018-11-26 | 2019-02-15 | 东南大学 | A kind of method of electrochemistry aptamer sensor detection aflatoxin B1 |
| CN109932409A (en) * | 2018-11-13 | 2019-06-25 | 重庆医科大学 | Renewable electrochemical immunosensor preparation method for sCD40L detection |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107543924A (en) * | 2017-09-06 | 2018-01-05 | 重庆医科大学 | Biology sensor detection myeloperoxidase based on copper palladium platinum mesh nanometer material |
| CN107621492A (en) * | 2017-09-06 | 2018-01-23 | 重庆医科大学 | Biology sensor preparation method for the detection of the sialylated glycans of α 2,3 |
| CN107621492B (en) * | 2017-09-06 | 2020-09-11 | 重庆医科大学 | Preparation method of biosensor for detecting alpha 2,3 sialylated glycan |
| CN108398477A (en) * | 2018-01-22 | 2018-08-14 | 昆明理工大学 | A kind of active electrochemical method of measurement phosphoric acid enol pyruvic acid carboxylase |
| CN108398477B (en) * | 2018-01-22 | 2020-08-25 | 昆明理工大学 | Electrochemical method for determining activity of phosphoenolpyruvate carboxylase |
| CN109932409A (en) * | 2018-11-13 | 2019-06-25 | 重庆医科大学 | Renewable electrochemical immunosensor preparation method for sCD40L detection |
| CN109342526A (en) * | 2018-11-26 | 2019-02-15 | 东南大学 | A kind of method of electrochemistry aptamer sensor detection aflatoxin B1 |
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Application publication date: 20170308 |