CN109932409A - Renewable electrochemical immunosensor preparation method for sCD40L detection - Google Patents
Renewable electrochemical immunosensor preparation method for sCD40L detection Download PDFInfo
- Publication number
- CN109932409A CN109932409A CN201811344505.4A CN201811344505A CN109932409A CN 109932409 A CN109932409 A CN 109932409A CN 201811344505 A CN201811344505 A CN 201811344505A CN 109932409 A CN109932409 A CN 109932409A
- Authority
- CN
- China
- Prior art keywords
- scd40l
- ultrapure water
- mwcnts
- pei
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 102400000432 CD40 ligand, soluble form Human genes 0.000 title 1
- 101800000267 CD40 ligand, soluble form Proteins 0.000 title 1
- 239000000463 material Substances 0.000 claims abstract description 17
- 239000010931 gold Substances 0.000 claims abstract description 5
- 229910052737 gold Inorganic materials 0.000 claims abstract description 5
- 239000002105 nanoparticle Substances 0.000 claims abstract description 5
- 239000003446 ligand Substances 0.000 claims abstract description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 26
- 239000012498 ultrapure water Substances 0.000 claims description 26
- 239000002048 multi walled nanotube Substances 0.000 claims description 15
- 229920002873 Polyethylenimine Polymers 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 9
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 5
- 238000001903 differential pulse voltammetry Methods 0.000 claims description 5
- 230000004048 modification Effects 0.000 claims description 4
- 238000012986 modification Methods 0.000 claims description 4
- 229910004042 HAuCl4 Inorganic materials 0.000 claims description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 3
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 3
- 229910052593 corundum Inorganic materials 0.000 claims description 3
- 238000005498 polishing Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 238000002604 ultrasonography Methods 0.000 claims description 3
- 229910001845 yogo sapphire Inorganic materials 0.000 claims description 3
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 102000009027 Albumins Human genes 0.000 claims 1
- 108010088751 Albumins Proteins 0.000 claims 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims 1
- -1 potassium ferricyanide Chemical compound 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 12
- 208000004476 Acute Coronary Syndrome Diseases 0.000 abstract description 6
- 239000000427 antigen Substances 0.000 abstract description 4
- 102000036639 antigens Human genes 0.000 abstract description 4
- 108091007433 antigens Proteins 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 230000001154 acute effect Effects 0.000 abstract description 3
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 208000011580 syndromic disease Diseases 0.000 abstract description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 2
- 210000004351 coronary vessel Anatomy 0.000 abstract description 2
- 238000000835 electrochemical detection Methods 0.000 abstract description 2
- 239000000090 biomarker Substances 0.000 abstract 1
- 229910052799 carbon Inorganic materials 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 10
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 8
- 230000005518 electrochemistry Effects 0.000 description 5
- 239000012472 biological sample Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 229960000935 dehydrated alcohol Drugs 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000012151 immunohistochemical method Methods 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 239000002114 nanocomposite Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 241000283725 Bos Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 239000003150 biochemical marker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000002041 carbon nanotube Substances 0.000 description 1
- 229910021393 carbon nanotube Inorganic materials 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000009131 signaling function Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Landscapes
- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
Abstract
The present invention relates to the preparation methods that can be used for predicting the renewable electrochemical immunosensor detected with the biomarker i.e. soluble CD 40 ligand (sCD40L) of diagnosing acute coronary syndrome, belong to technical field of electrochemical detection.It is characterized by: being used for immobilized sCD40L antibody as base material using multi-walled carbon nanotube-polyethyleneimine-gold nanoparticle nano-complex (c-MWCNTs-PEI-AuNPs) of carboxyl-functional first, it realizes the capture to sCD40L, and then quantitative detection is carried out to sCD40L.Since the preparation of c-MWCNTs-PEI-AuNPs nano-complex is simple, electric conductivity is good, with preferable stability and biggish specific surface area, it therefore can immobilized a large amount of antibody securely, and using the specific recognition of antibody and antigen, make the electrochemical immunosensor of building that there is stronger specificity.The present invention has high sensitivity, high specificity, convenient and efficient and reproducible advantage, and provides new method for the detection of sCD40L, provides useful information for dlinial prediction and diagnosis acute coronary artery syndrome.
Description
Technical field:
It is good and reliable biochemical for can be used as prediction and diagnosing acute coronary syndrome that the present invention relates to one kind
The preparation method and application of the electrochemical immunosensor of marker, that is, soluble CD 40 ligand (sCD40L) detection, is based on carboxyl
The multi-walled carbon nanotube (c-MWCNTs) of functionalization, polyethyleneimine (PEI) and gold nanoparticle (AuNPs) composite material it is straight
The reproducible immunosensor of direct type belongs to field of electrochemical detection for the detection of sCD40L.
Background technique:
Currently, cardiovascular disease threatens the health of the mankind, has become because its disease incidence, disability rate and lethality are higher
The main reason for leading to human death, wherein acute coronary syndrome is clinically common, serious cardiovascular disease.
SCD40L is a member in tumor necrosis factor superfamily, belongs to I type transmembrane glycoprotein, can promote the amplification of cell and move
It moves.Some researches show that the sCD40L in human serum can be used for acute coronary syndrome early diagnosis and predict its wind of falling ill
One reliable and good biochemical marker of danger.Therefore, the sCD40L in quantitative detection human serum is acute to predicting and diagnosing
Coronary syndrome is of great significance.
The traditional detection method of protein has immunocytochemical method (ICC), Immunohistochemical Method (IHC), Western Immuno
The methods of blot hybridization (WB), but these methods are there are many deficiencies, as sample handling processes are cumbersome, analysis time is longer,
Instrument or expensive reagents, sensitivity are more low, are not suitable for conventional clinical detection.In recent years, electrochemical immunosensor
It is concerned because of its convenient and efficient, sensitive the features such as, and is widely used to biochemical analysis, environmental monitoring, clinical research
With food quality detection etc. fields.
In the application of electrochemical immunosensor, in order to reach easy, quickly realize and detected to target substance
Purpose, suitable electrode modified material should be selected.In recent years, multi-walled carbon nanotube carbon nanotube (MWCNTs) is because its is excellent
The features such as electric conductivity, stronger adsorption capacity, good chemical stability and biggish specific surface area, it is widely used in electrochemistry
In immunosensor.But due to its π-pi-electron presence, the interaction of Van der Waals force is formed, there is stronger hydrophobicity, lead
It is caused to disperse in many solvents uneven, easy to reunite, so that it be made to receive many in the application of electrochemica biological sensor
Limitation.In order to improve the dispersibility of MWCNTs, its agglomeration is reduced, on the one hand selects c-MWCNTs because it has carboxyl, increases
Its dispersibility is added；On the other hand using the PEI of branching (quasi polymer for containing many amino in branch), have good
Water solubility is combined with c-MWCNTs, not only further improves the dispersibility of MWCNTs in a solvent, but also provided by it
A large amount of amino are that the further modification of material is laid a good foundation.Gold nanoparticle (AuNPs) has good electric conductivity, biggish
The features such as specific surface area, stronger adsorption capacity, good biocompatibility, has amplification electrochemical immunosensor electric signal
Function, the sensitivity of electrochemical immunosensor can be further increased, answered extensively in electrochemical immunosensor
With.The present invention utilizes the property of above-mentioned material, and electric signal not only is further amplified, but also passes through AuNPs and sCD40L antibody
Form Au-NH2Key, thus fixed sCD40L antibody, and it is able to maintain the activity of this antibody, sCD40L is quantified to realize
Detection.Since base material c-MWCNTs-PEI-AuNPs nanocomposite electrochemical properties are stablized, with sCD40L antibody
It is firmly combined, in alkaline solution, antibody antigen dissociation, and the connection of antibody and electrode surface is unaffected, exempts from electrochemistry
Epidemic disease sensor is used multiple times with reproducibility.
It is base material the present invention is based on c-MWCNTs-PEI-AuNPs nano-complex, establishes a kind of for biological sample
The preparation method of the renewable electrochemical immunosensor of Direct-type of middle sCD40L detection, is the sCD40L's in Organism Samples
Easily and fast quantitative detection provides a new method, provides accordingly for the prediction and diagnosis of clinically acute coronary artery syndrome
With reference to.
Summary of the invention:
The object of the present invention is to provide a kind of electrochemistry immuno-sensings for the sCD40L quantitative detection in biological sample
The preparation method of device, feature the following steps are included:
(1) carboxyl function multi-walled carbon nano-tube (c-MWCNTs)-polyethyleneimine (PEI)-gold nanoparticle (AuNPs)
The preparation of base material；
(2) electro-chemistry immunity biosensor is established, sCD40L is measured, draws standard curve.
The preparation process of c-MWCNTs-PEI-AuNPs nano-complex of the present invention, feature the following steps are included:
In the ultrapure water for weighing 2mg c-MWCNTs to 2mL, ultrasonic 1-3h makes it be uniformly dispersed.Under constant stirring plus
Enter suitable n-hydroxysuccinimide (NHS) and 1- ethyl -3- (3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution
Its carboxyl is activated, 100 μ L PEI solution are added after stirring 30min and continue to stir 2-5h.After above-mentioned material washing repeatedly, point
It is dispersed in 2mL ultrapure water, is successively separately added into 100 μ L HAuCl4·6H2The NaBH of O (1%) and 900 μ L (30mM)4Solution stirs
It mixes overnight, is dispersed in 2mL ultrapure water after washing repeatedly, c-MWCNTs-PEI-AuNPs nano-complex can be obtained, by it
Be dispersed in 2mL ultrapure water be stored in it is spare in 4 DEG C of refrigerators.
The heretofore described sCD40L concentration established electrochemical immunosensor, measure in biological sample, draws mark
Directrix curve, it is characterised in that the following steps are included:
(1) respectively with 0.3 μm and 0.05 μm of Al2O3Powder is by polishing electrode to mirror surface, then respectively with appropriate ultrapure
By said sequence by electrode each 5min of ultrasound, drying at room temperature is spare for water, dehydrated alcohol, ultrapure water；
(2) the c-MWCNTs-PEI-AuNPs nano-complex for preparing 6 μ L is added dropwise in electrode surface, drying at room temperature；
(3) electrode surface of the sCD40L antibody dropwise addition of 6 μ L after modification is placed in 4 DEG C of refrigerators and is incubated for 10h；
(4) after the unbonded firm sCD40L antibody of the electrode surface after incubation being washed away with ultrapure water, be added dropwise 6 μ L oxen
Seralbumin (BSA, 0.25%) solution is incubated at room temperature 30min；
(5) after being washed away the extra BSA of electrode surface with ultrapure water, the sCD40L of 6 μ L various concentrations is added dropwise respectively
Electrode surface is incubated for 45min；
(6) after not washed away with the sCD40L of sCD40L antibody firm connection with ultrapure water, electrode is placed in the iron of 5mM
Potassium cyanide solution (5mM K3[Fe(CN)6]、5mM K4[Fe(CN)6], 0.1M KCl) in characterized, use differential pulse voltammetry
Method (DPV) measures its current-responsive value.
(7) in a linear relationship according to gained peak current difference and the logarithm of sCD40L concentration, draw working curve.
Compared with prior art, the present invention is a kind of electro-chemistry immunity biography for sCD40L quantitative detection in biological sample
The preparation method of sensor, the feature protruded is:
(1) system of electrochemical immunosensor is introduced into using c-MWCNTs-PEI-AuNPs nanocomposite as substrate
In standby, electric conductivity is not only increased, electron transmission is accelerated, and increases the supported quantity of biomolecule, and then improve electricity
The sensitivity of chemo-immunity sensor and biocompatibility；
(2) system of the electrochemical immunosensor of this method preparation due to its base material (c-MWCNTs-PEI-AuNPs)
Standby process is simple, convenient and electrochemical properties are stablized, and antibody is firmly combined with base material.Although under alkaline condition, antibody
It is dissociated with antigen, and the combination of antibody and base material is not affected substantially, therefore has excellent stability, reproducibility and again
Natural disposition；
(3) electrochemical immunosensor of this method preparation to the prevention of acute coronary syndrome and can examine for clinic
It is disconnected that effective information is provided, facilitate the diagnosis and prevention of acute coronary syndrome.
(4) electrochemical immunosensor of this method preparation is due to having using the specific binding between antibody antigen
Good specificity, preparation process is simple, detecting step is less, detects fast speed, commercialization is easy to implement, to promote
The development of translational medicine.
Detailed description of the invention:
Fig. 1 is the building schematic diagram of electrochemical immunosensor in the present invention.
Fig. 2 is that the transmission electron microscope figures of different synthesis steps, energy spectrum diagram of base material and ultraviolet in the present invention-can
See abosrption spectrogram.
Fig. 3 be the DPV curve that is obtained when detecting soluble CD 40 ligand of electrochemical immunosensor of the invention and its
The linear relationship of peak current difference and log concentration.
Specific embodiment:
The present invention is further elaborated combined with specific embodiments below, it should be appreciated that these embodiments are merely to illustrate
The present invention rather than limit the scope of the invention.
Embodiment 1
Step 1. weighs in the ultrapure water of 2mg c-MWCNTs to 2mL, and ultrasonic 1-3h makes it be uniformly dispersed.Constantly stirring
It mixes the lower suitable EDC and NHS solution of addition and activates its carboxyl, 100 μ L PEI solution are added after magnetic agitation 30min and continue to stir
Mix 2-5h.After above-mentioned material washing repeatedly, it is dispersed in 2mL ultrapure water, is successively separately added into 100 μ L HAuCl4·6H2O
(1%) and the NaBH of 900 μ L4(30mM) is stirred overnight, and is dispersed in 2mL ultrapure water after washing repeatedly, c- can be obtained
MWCNTs-PEI-AuNPs nano-complex, be dispersed in 2mL ultrapure water be stored in it is spare in 4 DEG C of refrigerators；
Step 2. is respectively with 0.3 μm and 0.05 μm of Al2O3Powder is by polishing electrode to mirror surface, then respectively with appropriate super
In the above sequence by each ultrasound 5min of electrode, drying at room temperature is spare for pure water, dehydrated alcohol, ultrapure water；
Step 3. takes the c-MWCNTs-PEI-AuNPs nano-complex of the above-mentioned 6 μ L prepared to be added dropwise in electrode surface,
Drying at room temperature；
After the electrode surface after modification is added dropwise in the sCD40L antibody of 6 μ L by step 4., it is placed in 4 DEG C of refrigerators and is incubated for 10h；
After step 5. is washed away the sCD40L antibody of the electrode surface after incubation not being firmly combined with ultrapure water, 6 μ are added dropwise
L BSA (0.25%) solution is incubated at room temperature 30min；
After step 6. is washed away the extra BSA of the electrode surface after incubation with ultrapure water, 6 μ L are added dropwise respectively on the electrode not
SCD40L with concentration is placed in 37 DEG C of incubation 45min；
After step 7. will not washed away with the sCD40L of sCD40L antibody firm connection with ultrapure water, iron cyaniding is placed it in
Potassium solution (5mM K3[Fe(CN)6]、5mM K4[Fe(CN)6], 0.1M KCl) in characterized, measure its current-responsive with DPV
Value；
Step 8. is in a linear relationship according to the logarithm that gained peak point current acquires its difference and sCD40L concentration, draws work
Curve；Measurement result shows sCD40L concentration in 10fg mL-1-100pg mL-1It is linear in range, linearly dependent coefficient
Square (R2) it is 0.99, detection is limited to 3fg mL-1(S/N=3)；
The present invention is used to detect the interfering substance in sCD40L and blood plasma by step 9., the results showed that the interference in blood plasma
Electric current analog value of the current-responsive value of substance well below sCD40L illustrates specific good, the strong antijamming capability of sensor,
It can exclude the interference of other substances；
The sensor in the present invention is placed in 4 DEG C of preservations in refrigerator by step 10., and discontinuity detection sensor current is rung
It answers, current-responsive is still the 89.53% of initial current after storage 28 days, shows that sensor is with good stability；
5 electrochemical immunosensors of the invention are used to detect same concentration (1pg mL by step 11.-1)
SCD40L, relative standard deviation 1.38% show that this sensor has good reproducibility；
The present invention is used to detect same concentration (1pg mL by step 12.-1) sCD40L after, be put into dissociation solution (30mM
NaOH it takes out, repeats the above steps after carefully being rinsed with ultrapure water, the results showed that it is repeated 5 times current value after rinse 60s in)
Still it is the 95.79% of initial current, shows that this sensor is good since the firm connection of the stabilization of material, antibody and material has
Reproducibility.
Claims (3)
1. renewable electrochemical immunosensor preparation method of the one kind for soluble CD 40 ligand (sCD40L) detection, special
Sign be the following steps are included:
(1) carboxyl function multi-walled carbon nano-tube (c-MWCNTs)-polyethyleneimine (PEI)-gold nanoparticle (AuNPs) substrate
The preparation of material；
(2) electrochemical immunosensor is established, sCD40L is measured, draws standard curve.
2. the preparation process of c-MWCNTs-PEI-AuNPs nano-complex specifically includes following step according to claim 1
Suddenly, it is characterised in that the following steps are included:
It weighs in the ultrapure water of c-MWCNTs to the 2mL of 2mg, ultrasonic 1-3h makes it be uniformly dispersed.N- is added under constant stirring
HOSu NHS (NHS) and 1- ethyl -3- (3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution activate its carboxylic
Base, 100 μ L PEI solution, which are added, in stirring 30min later continues to stir 2-5h.After above-mentioned material washing repeatedly, it is dispersed in
In 2mL ultrapure water, it is successively separately added into 100 μ L HAuCl4·6H2The NaBH of O (1%) and 900 μ L (30mM)4Solution is stirred
Night is dispersed in 2mL ultrapure water after washing repeatedly, c-MWCNTs-PEI-AuNPs nano-complex can be obtained, dispersed
It is stored in 2mL ultrapure water spare in 4 DEG C of refrigerators.
3. according to claim 1 establish renewable electrochemical immunosensor, sCD40L is measured, draws standard curve,
Characterized by the following steps:
(1) respectively with 0.3 μm and 0.05 μm of Al2O3Powder is by polishing electrode to mirror surface, then respectively with appropriate ultrapure water, nothing
By said sequence by each ultrasound 5min of electrode, drying at room temperature is spare for water-ethanol, ultrapure water；
(2) the c-MWCNTs-PEI-AuNPs nano-complex for preparing 6 μ L is added dropwise in electrode surface, drying at room temperature；
(3) it after the electrode surface after modification being added dropwise in the sCD40L antibody of 6 μ L, is placed in 4 DEG C of refrigerators and is incubated for 10h；
(4) after the sCD40L antibody of the electrode surface after incubation not being firmly combined being washed away with ultrapure water, be added dropwise 6 μ L cow's serums
Albumin (BSA, 0.25%) solution is incubated at room temperature 30min；
(5) after being washed away the extra BSA of the electrode surface after incubation with ultrapure water, the sCD40L of 6 μ L various concentrations is dripped respectively
It is added in electrode surface and is incubated for 45min；
(6) after not washed away with the sCD40L of sCD40L antibody firm connection with ultrapure water, it is placed in the potassium ferricyanide solution of 5mM
(5mM K3[Fe(CN)6]、5mM K4[Fe(CN)6], 0.1M KCl) in characterized, with differential pulse voltammetry (DPV) measure
Its current-responsive value；
(7) in a linear relationship according to gained peak current difference and the logarithm of sCD40L concentration, draw working curve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811344505.4A CN109932409A (en) | 2018-11-13 | 2018-11-13 | Renewable electrochemical immunosensor preparation method for sCD40L detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811344505.4A CN109932409A (en) | 2018-11-13 | 2018-11-13 | Renewable electrochemical immunosensor preparation method for sCD40L detection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109932409A true CN109932409A (en) | 2019-06-25 |
Family
ID=66984618
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811344505.4A Pending CN109932409A (en) | 2018-11-13 | 2018-11-13 | Renewable electrochemical immunosensor preparation method for sCD40L detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109932409A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110927238A (en) * | 2019-12-12 | 2020-03-27 | 山东理工大学 | Preparation method and application of sandwich type photoelectrochemical sensor for detecting prostate specific antigen |
| CN111426849A (en) * | 2020-04-13 | 2020-07-17 | 云南万魁生物科技有限公司 | Method for determining 14-3-3 protein expression level in soluble total protein |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106483281A (en) * | 2016-09-26 | 2017-03-08 | 重庆医科大学 | Renewable electrochemical immunosensor preparation method for sCD40L detection |
| CN107389949A (en) * | 2017-09-06 | 2017-11-24 | 重庆医科大学 | A kind of electrochemical immunosensor preparation method for PCSK9 Protein Detections |
-
2018
- 2018-11-13 CN CN201811344505.4A patent/CN109932409A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106483281A (en) * | 2016-09-26 | 2017-03-08 | 重庆医科大学 | Renewable electrochemical immunosensor preparation method for sCD40L detection |
| CN107389949A (en) * | 2017-09-06 | 2017-11-24 | 重庆医科大学 | A kind of electrochemical immunosensor preparation method for PCSK9 Protein Detections |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110927238A (en) * | 2019-12-12 | 2020-03-27 | 山东理工大学 | Preparation method and application of sandwich type photoelectrochemical sensor for detecting prostate specific antigen |
| CN110927238B (en) * | 2019-12-12 | 2022-10-11 | 山东理工大学 | Preparation method and application of sandwich type photoelectrochemical sensor for detecting prostate specific antigen |
| CN111426849A (en) * | 2020-04-13 | 2020-07-17 | 云南万魁生物科技有限公司 | Method for determining 14-3-3 protein expression level in soluble total protein |
| CN111426849B (en) * | 2020-04-13 | 2024-02-09 | 云南万魁生物科技有限公司 | Method for measuring 14-3-3 protein expression level in soluble total protein |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jia et al. | Triple signal amplification using gold nanoparticles, bienzyme and platinum nanoparticles functionalized graphene as enhancers for simultaneous multiple electrochemical immunoassay | |
| Tang et al. | A novel cytosensor based on Pt@ Ag nanoflowers and AuNPs/Acetylene black for ultrasensitive and highly specific detection of Circulating Tumor Cells | |
| Li et al. | Simultaneous electrochemical immunoassay of three liver cancer biomarkers using distinguishable redox probes as signal tags and gold nanoparticles coated carbon nanotubes as signal enhancers | |
| Yang et al. | Ultrasensitive electrochemical immunosensor for carbohydrate antigen 19-9 using Au/porous graphene nanocomposites as platform and Au@ Pd core/shell bimetallic functionalized graphene nanocomposites as signal enhancers | |
| Zhu et al. | Amperometric immunosensor for simultaneous detection of three analytes in one interface using dual functionalized graphene sheets integrated with redox-probes as tracer matrixes | |
| CN106442994B (en) | A kind of preparation method and application of the electrochemical immunosensor based on Ag@Au nano composite materials | |
| Li et al. | An ultrasensitive electrochemical immunosensor for CEA using MWCNT-NH 2 supported PdPt nanocages as labels for signal amplification | |
| Liu et al. | Ultrasensitive electrochemical immunosensor for alpha fetoprotein detection based on platinum nanoparticles anchored on cobalt oxide/graphene nanosheets for signal amplification | |
| Lv et al. | The label-free immunosensor based on rhodium@ palladium nanodendrites/sulfo group functionalized multi-walled carbon nanotubes for the sensitive analysis of carcino embryonic antigen | |
| Zhang et al. | GoldMag nanocomposite-functionalized graphene sensing platform for one-step electrochemical immunoassay of alpha-fetoprotein | |
| Huang et al. | Highly sensitive luminol electrochemiluminescence immunosensor based on platinum-gold alloy hybrid functionalized zinc oxide nanocomposites for catalytic amplification | |
| Ji et al. | Ultrasensitive sandwich-type electrochemical immunosensor based on a novel signal amplification strategy using highly loaded palladium nanoparticles/carbon decorated magnetic microspheres as signal labels | |
| Feng et al. | Recent advances of carbon nanotubes‐based electrochemical immunosensors for the detection of protein cancer biomarkers | |
| Zhu et al. | Simultaneous detection of four biomarkers with one sensing surface based on redox probe tagging strategy | |
| Wang et al. | A supersandwich multienzyme–DNA label based electrochemical immunosensor | |
| Zhou et al. | Carbon nanospheres-promoted electrochemical immunoassay coupled with hollow platinum nanolabels for sensitivity enhancement | |
| Zhang et al. | Signal-on electrochemiluminescent immunosensor based on poly (amidoamine) dendrimer functionalized carbon nanodots amplification for ultrasensitive detection of α-fetoprotein | |
| Jiang et al. | Self-enhanced N-(aminobutyl)-N-(ethylisoluminol) derivative-based electrochemiluminescence immunosensor for sensitive laminin detection using PdIr cubes as a mimic peroxidase | |
| CN109613244B (en) | Preparation method and application of Ag @ Pt-CuS labeled immunosensor | |
| Dong et al. | “Gold-plated” IRMOF-3 and sea cucumber-like Pd@ PtRh SNRs based sandwich-type immunosensor for dual-mode detection of PCT | |
| CN105628931A (en) | Preparation and application of alpha fetoprotein electrochemical immunosensor based on silver deposition | |
| CN106483281A (en) | Renewable electrochemical immunosensor preparation method for sCD40L detection | |
| CN106198699B (en) | Prepare two kinds of secondary antibody conjugates and its at the same the method that detects alpha-fetoprotein and carcinomebryonic antigen | |
| CN108802390A (en) | A kind of preparation of the pancreatic tumour marker immunosensor based on graphene-gold-palladium nanocomposite | |
| CN106093396A (en) | A kind of preparation method and application of immunosensor based on Au GQD@PtPd |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190625 |