CN106483281A - Renewable electrochemical immunosensor preparation method for sCD40L detection - Google Patents

Abstract

The invention relates to a preparation method of a reproducible electrochemical immunosensor for the detection of soluble CD40 ligand (sCD40L), which is a biomarker for predicting and diagnosing acute coronary syndrome, and belongs to the technical field of electrochemical detection. It is characterized in that: firstly, carboxyl-functionalized multi-walled carbon nanotube-polyethyleneimine-gold nanoparticle nanocomposite (c-MWCNTs-PEI-AuNPs) is used as the substrate material to immobilize sCD40L antibody, so as to realize the anti-sCD40L Capture, and then quantitatively detect sCD40L. Due to the simple preparation of c‑MWCNTs‑PEI‑AuNPs nanocomposites, good electrical conductivity, good stability and large specific surface area, it can firmly immobilize a large number of antibodies, and utilize the specific recognition of antibodies and antigens , so that the constructed electrochemical immunosensor has strong specificity. The invention has the advantages of high sensitivity, strong specificity, convenience, quickness and repeated use, provides a new method for detecting sCD40L, and provides useful information for clinical prediction and diagnosis of acute coronary syndrome.

Description

Preparation method of regenerative electrochemical immunosensor for sCD40L detection

Technical field:

The present invention relates to a method for the preparation of an electrochemical immunosensor for the detection of soluble CD40 ligand (sCD40L), which can be used as a good and reliable biochemical marker for prediction and diagnosis of acute coronary syndrome, based on carboxyl-functionalized multi-walled carbon Direct regenerative immunosensors of nanotubes (c-MWCNTs), polyethyleneimine (PEI), and gold nanoparticles (AuNPs) nanocomposites for the detection of sCD40L belong to the field of electrochemical detection.

Background technique:

At present, cardiovascular disease has become a major cause of human death due to its high morbidity, disability and mortality, threatening human health, and acute coronary syndrome is a common and serious cardiovascular disease in clinical practice. . sCD40L is a member of the tumor necrosis factor superfamily and belongs to type I transmembrane glycoprotein, which can promote cell expansion and migration. Studies have shown that sCD40L in human serum can be used as a reliable and good biochemical marker for early diagnosis of acute coronary syndrome and prediction of its risk. Therefore, the quantitative detection of sCD40L in human serum has very important significance for the prediction and diagnosis of acute coronary syndrome.

Traditional protein detection methods include immunocytochemistry (ICC), immunohistochemistry (IHC), Western blot hybridization (WB) and other methods, but these methods have many shortcomings, such as cumbersome sample processing and long analysis time , expensive instruments or reagents, low sensitivity, etc., are not suitable for routine clinical testing. In recent years, electrochemical immunosensors have attracted much attention due to their convenience, quickness, and sensitivity, and have been widely used in biochemical analysis, environmental monitoring, clinical research, and food quality testing.

In the application of electrochemical immunosensors, in order to achieve the purpose of simple and rapid detection of target substances, appropriate electrode modification materials should be selected. In recent years, multi-walled carbon nanotubes (MWCNTs) have been widely used in electrochemical immunosensors due to their excellent electrical conductivity, strong adsorption capacity, good electrochemical stability, and large specific surface area. . However, due to the existence of its π-π electrons, it forms the interaction of van der Waals force and has strong hydrophobicity, which leads to its uneven dispersion and easy aggregation in many solvents, so its application in electrochemical immunosensors is subject to many limit. In order to improve the dispersion of MWCNTs and reduce their agglomeration, on the one hand, c-MWCNTs is selected because of its carboxyl group, which increases its dispersion; on the other hand, branched PEI (a type of polymer containing many amino groups in the branch) is used. , has good water solubility, combined with c-MWCNTs, not only further improves the dispersibility of MWCNTs in solvents, but also provides a large number of amino groups to lay the foundation for further modification of materials. Gold nanoparticles (AuNPs) have the characteristics of good electrical conductivity, large specific surface area, strong adsorption capacity, and good biocompatibility. The sensitivity of the sensor has been widely used in electrochemical immunosensors. The present invention not only further amplifies the electrical signal by utilizing the properties of the above materials, but also forms Au-NH ₂ bonds between AuNPs and sCD40L antibody to immobilize the sCD40L antibody and maintain the activity of the antibody, thereby realizing the quantitative detection of sCD40L. Due to the stable electrochemical properties of the substrate material c-MWCNTs-PEI-AuNPs nanocomposite, it binds firmly to the sCD40L antibody. In alkaline solution, the antibody antigen dissociates, while the connection between the antibody and the electrode surface is not affected. Immunosensors are reproducible and can be reused many times.

Based on the c-MWCNTs-PEI-AuNPs nanocomposite as the base material, the present invention establishes a method for preparing a direct-type reproducible electrochemical immunosensor for the detection of sCD40L in biological samples, which is convenient and convenient for sCD40L in biological samples. Rapid quantitative detection provides a new method and provides a reference for the prediction and diagnosis of acute coronary syndrome in clinical practice.

Invention content:

The object of the present invention is to provide a kind of preparation method of the renewable electrochemical immunosensor that is used for the quantitative detection of sCD40L in the biological sample, and its feature comprises the following steps:

(1) Preparation of carboxyl-functionalized multi-walled carbon nanotubes (c-MWCNTs)-polyethyleneimine (PEI)-gold nanoparticles (AuNPs) substrate material;

(2) Establish a regenerative electrochemical immunological biosensor, measure sCD40L, and draw a standard curve.

The preparation process of the c-MWCNTs-PEI-AuNPs nanocomposite of the present invention is characterized in that it comprises the following steps:

Weigh 2mg of c-MWCNTs into 2mL of ultrapure water, and sonicate for 1-3h to disperse evenly. Add appropriate amount of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution under constant stirring to activate its carboxyl group, stirring After 30 min, 100 μL of PEI solution was added and stirring continued for 2-5 h. After the above materials were washed several times, they were dispersed in 2 mL ultrapure water, and 100 μL HAuCl ₄ ·6H ₂ O (1%) and 900 μL (30 mM) NaBH ₄ solutions were added successively and stirred overnight, and then dispersed in 2 mL ultrapure water after washing several times. c-MWCNTs-PEI-AuNPs nanocomposite can be obtained in pure water, which is dispersed in 2mL ultrapure water and stored in a 4°C refrigerator for later use.

The establishment of the renewable electrochemical immunosensor described in the present invention, the determination of the sCD40L concentration in the biological sample, and the drawing of a standard curve are characterized in that it includes the following steps:

(1) Polish the electrode to the mirror surface with 0.3 μm and 0.05 μm Al ₂ O ₃ powder respectively, then use appropriate amount of ultrapure water, absolute ethanol, and ultrapure water to ultrasonically clean the electrode for 5 minutes each in the above sequence, and dry it at room temperature for later use ;

(2) Add 6 μL of the prepared c-MWCNTs-PEI-AuNPs nanocomposite dropwise on the electrode surface and dry at room temperature;

(3) Add 6 μL of sCD40L antibody dropwise on the surface of the modified electrode and incubate in a refrigerator at 4°C for 10 h;

(4) After the unbound sCD40L antibody on the surface of the incubated electrode was washed away with ultrapure water, 6 μL of bovine serum albumin (BSA, 0.25%) solution was added dropwise and incubated at room temperature for 30 minutes;

(5) After washing away excess BSA on the surface of the electrode with ultrapure water, add 6 μL of sCD40L of different concentrations to the surface of the electrode and incubate for 45 minutes;

(6) After washing away the sCD40L not bound to the sCD40L antibody with ultrapure water, place the electrode in 5mM potassium ferricyanide solution (5mM K ₃ [Fe(CN) ₆ ], 5mM K ₄ [Fe(CN) ₆ ], 0.1M KCl), and use differential pulse voltammetry (DPV) to measure its current response value;

(7) According to the linear relationship between the obtained peak current difference and the logarithm of the sCD40L concentration, draw a working curve;

(8) Rinse the electrode that has detected sCD40L in dissociation solution (30mM NaOH) for 60s, take it out, carefully rinse it with ultrapure water, and store it in a refrigerator at 4°C for future use.

Compared with the prior art, the present invention is a preparation method of a renewable electrochemical immunosensor for quantitative detection of sCD40L in biological samples, and its prominent features are:

(1) Introducing c-MWCNTs-PEI-AuNPs nanocomposites as a substrate into the preparation of electrochemical immunosensors not only enhances the electrical conductivity, accelerates electron transfer, but also increases the immobilization capacity of biomolecules, thereby improving the Sensitivity of electrochemical immunosensors.

(2) The regenerative electrochemical immunosensor prepared by this method is simple, convenient and stable in electrochemical properties due to the simple, convenient and stable electrochemical properties of the substrate material (c-MWCNTs-PEI-AuNPs), and the antibody is firmly combined with the substrate material. Under alkaline conditions, the antibody and the antigen dissociate, but the binding of the antibody to the base material is not greatly affected, so it has excellent stability, reproducibility and reproducibility, and can be used repeatedly.

(3) The regenerative electrochemical immunosensor prepared by this method can provide effective information for clinical prevention and diagnosis of acute coronary syndrome, and is helpful for the diagnosis and prevention of acute coronary syndrome.

(4) The regenerative electrochemical immunosensor prepared by this method has good specificity due to the use of specific binding between antibody and antigen, and its preparation process is simple, with fewer detection steps and faster detection speed, which is convenient for commercialization. To promote the development of translational medicine.

Description of drawings:

Figure 1 is a schematic diagram of the construction of the regenerative electrochemical immunosensor in the present invention.

Fig. 2 is a transmission electron microscope image, an energy spectrum image and an ultraviolet-visible absorption spectrum image of different synthesis steps of the base material in the present invention.

Fig. 3 is the DPV curve obtained when the regenerative electrochemical immunosensor of the present invention detects soluble CD40 ligand and the linear relationship between the peak current difference and the logarithm of the concentration.

detailed description:

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1

Step 1. Weigh 2mg of c-MWCNTs into 2mL of ultrapure water and sonicate for 1-3h to disperse evenly. Add appropriate amount of EDC and NHS solution to activate its carboxyl group under constant stirring, add 100 μL PEI solution after magnetic stirring for 30 minutes and continue stirring for 2-5 hours. After washing the above materials several times, disperse in 2mL ultrapure water, add 100μL HAuCl ₄ 6H ₂ O (1%) and 900μL NaBH ₄ (30mM) successively and stir overnight, wash several times and disperse in 2mL ultrapure water In water, the c-MWCNTs-PEI-AuNPs nanocomposite can be obtained, which is dispersed in 2mL ultrapure water and stored in a 4°C refrigerator for later use;

Step 2. Use 0.3μm and 0.05μm Al ₂ O ₃ powder to polish the electrode to the mirror surface, and then use appropriate amount of ultrapure water, absolute ethanol, and ultrapure water to ultrasonically clean the electrodes for 5 minutes in the above order, and dry them at room temperature for later use ;

Step 3. Take 6 μL of the prepared c-MWCNTs-PEI-AuNPs nanocomposite dropwise on the surface of the electrode, and dry at room temperature;

Step 4. After dropping 6 μL of sCD40L antibody onto the surface of the modified electrode, place it in a 4°C refrigerator and incubate for 10 h;

Step 5. After washing away the unbound sCD40L antibody on the surface of the incubated electrode with ultrapure water, add 6 μL of BSA (0.25%) solution dropwise and incubate at room temperature for 30 minutes;

Step 6. After washing away excess BSA on the surface of the incubated electrode with ultrapure water, add 6 μL of different concentrations of sCD40L to the electrode and incubate at 37°C for 45 minutes;

Step 7. After washing away the sCD40L not bound to the sCD40L antibody with ultrapure water, place it in a potassium ferricyanide solution (5mM K ₃ [Fe(CN) ₆ ], 5mM K ₄ [Fe(CN) ₆ ] , 0.1M KCl), and use DPV to measure its current response value;

Step 8. According to the obtained peak current value, the logarithm of the difference and the sCD40L concentration is linear, and the working curve is drawn; the measurement results show that the sCD40L concentration is linear in the range of 10fg mL ^- 1-100pg mL ^-1 , and the linear correlation The coefficient square (R ² ) is 0.99, and the detection limit is 3fg mL ^-1 (S/N=3);

Step 9. The present invention is used to detect sCD40L and interfering substances in plasma, and the results show that the current response value of interfering substances in plasma is far lower than that of sCD40L, indicating that the sensor has good specificity and strong anti-interference ability. Ability to exclude interference from other substances;

Step 10. Store the above sensor in the present invention at 4°C in a refrigerator, and detect the current response of the sensor intermittently. After 28 days of storage, the current response is still 89.53% of the initial current, indicating that the sensor has good stability;

Step 11. Using five electrochemical immunosensors of the present invention to detect sCD40L at the same concentration (1 pg mL ^-1 ), the relative standard deviation is 1.38%, indicating that the sensor has good reproducibility;

Step 12. After using the present invention to detect sCD40L at the same concentration (1pg mL ^-1 ), put it into the dissociation solution (30mMNaOH) for rinsing for 60s, take it out, rinse carefully with ultrapure water, and repeat the above steps. The results show that The current value was still 95.79% of the initial current after repeated 5 times, indicating that the sensor has good reproducibility due to the stability of the material and the firm combination of the antibody and the material, and can be used repeatedly.

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.