CN109932409A - Preparation method of regenerable electrochemical immunosensor for sCD40L detection - Google Patents

Abstract

The invention relates to a preparation method of a regenerable electrochemical immunosensor which can be used for predicting and diagnosing a biomarker of acute coronary syndrome, namely soluble CD40 ligand (sCD40L) detection, 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 a base material for immobilizing sCD40L antibody, so as to realize the anti-sCD40L antibody. Capture, and then quantitatively detect sCD40L. Because the c-MWCNTs-PEI-AuNPs nanocomposite is simple to prepare, has 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 reproducibility, provides a new method for the detection of sCD40L, and provides useful information for clinical prediction and diagnosis of acute coronary syndrome.

Description

Preparation method of regenerable electrochemical immunosensor for sCD40L detection

Technical field:

The invention relates to a preparation method and application of an electrochemical immunosensor for detection of soluble CD40 ligand (sCD40L), which can be used as a good and reliable biochemical marker for predicting and diagnosing acute coronary syndrome. A direct-type regenerable immunosensor of composite materials of walled carbon nanotubes (c-MWCNTs), polyethyleneimine (PEI) and gold nanoparticles (AuNPs) is used for the detection of sCD40L and belongs to the field of electrochemical detection.

Background technique:

At present, cardiovascular disease has become the main cause of human death because of its high morbidity, morbidity and mortality, threatening human health, and acute coronary syndrome is a common clinical and serious cardiovascular disease. . sCD40L, a member of the tumor necrosis factor superfamily, is a type I transmembrane glycoprotein that promotes cell expansion and migration. Studies have shown that sCD40L in human serum can be a reliable and good biochemical marker for early diagnosis of acute coronary syndrome and prediction of its risk. Therefore, quantitative detection of sCD40L in human serum is of great significance for the prediction and diagnosis of acute coronary syndrome.

Traditional protein detection methods include immunocytochemistry (ICC), immunohistochemistry (IHC), western blotting (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., not suitable for routine clinical testing. In recent years, electrochemical immunosensors have attracted much attention due to their convenience, speed, 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 and carbon nanotubes (MWCNTs) have been widely used in electrochemical immunosensors due to their excellent electrical conductivity, strong adsorption capacity, good chemical stability, and large specific surface area. However, due to the existence of its π-π electrons, the interaction of van der Waals force is formed, and it has strong hydrophobicity, which leads to its uneven dispersion and easy agglomeration in many solvents, so its application in electrochemical biosensors has received a lot of attention. limit. In order to improve the dispersibility of MWCNTs and reduce their agglomeration, on the one hand, c-MWCNTs are selected because of their carboxyl groups, which increases their dispersibility; on the other hand, branched PEI (a class of polymers containing many amino groups in the branches) is used. , with 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 for further modification of the material. Gold nanoparticles (AuNPs) have the characteristics of good electrical conductivity, large specific surface area, strong adsorption capacity, good biocompatibility, etc., and have the function of amplifying the electrical signal of electrochemical immunosensors, which can further improve the electrochemical immunity. The sensitivity of the sensor is widely used in electrochemical immunosensors. The present invention utilizes the properties of the above-mentioned materials, not only further amplifies the electrical signal, but also forms Au-NH ₂ bonds through AuNPs and the sCD40L antibody, thereby immobilizing the sCD40L antibody, and maintaining 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 an alkaline solution, the antibody antigen dissociates, while the connection between the antibody and the electrode surface is not affected, making the electrochemical Immunosensors are regenerative and can be used multiple times.

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

Invention content:

The object of the present invention is to provide a preparation method of an electrochemical immunosensor for quantitative detection of sCD40L in biological samples, which is characterized by comprising the following steps:

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

(2) Establish an electrochemical immunobiosensor, measure sCD40L, and draw a standard curve.

The preparation process of the c-MWCNTs-PEI-AuNPs nanocomposite according to the present invention is characterized by comprising the following steps:

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

The establishment of an 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 by comprising the following steps:

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

(2) 6 μL of the prepared c-MWCNTs-PEI-AuNPs nanocomposite was dropped on the electrode surface and dried at room temperature;

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

(4) After washing off the unbound sCD40L antibody on the surface of the electrode after incubation 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 flushing the excess BSA on the electrode surface with ultrapure water, drop 6 μL of sCD40L with different concentrations on the electrode surface and incubate for 45min;

(6) After the sCD40L that was not firmly bound to the sCD40L antibody was washed away with ultrapure water, the electrode was placed in a 5 mM potassium ferricyanide solution (5 mM K ₃ [Fe(CN) ₆ ], 5 mM K ₄ [Fe(CN) ) ₆ ], 0.1M KCl), and its current response value was measured by differential pulse voltammetry (DPV).

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

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

(1) The c-MWCNTs-PEI-AuNPs nanocomposite was introduced as a substrate into the preparation of electrochemical immunosensors, which not only enhanced the electrical conductivity, accelerated the electron transfer, but also increased the immobilized capacity of biomolecules, thereby improving the Sensitivity and biocompatibility of electrochemical immunosensors;

(2) The electrochemical immunosensor prepared by this method has a simple and convenient preparation process and stable electrochemical properties of its substrate material (c-MWCNTs-PEI-AuNPs), and the antibody is firmly bound to the substrate material. Although under alkaline conditions, the antibody dissociates from the antigen, and the binding of the antibody to the substrate material is not greatly affected, so it has excellent stability, reproducibility and regeneration;

(3) The electrochemical immunosensor prepared by the 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 electrochemical immunosensor prepared by this method has good specificity due to the specific binding between the antibody and antigen. The development of translational medicine.

Description of drawings:

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

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 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 ways:

The present invention will be further described below with reference to specific embodiments, and it should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

Example 1

Step 1. Weigh 2 mg of c-MWCNTs into 2 mL of ultrapure water, and sonicate for 1-3 h to disperse evenly. Under constant stirring, an appropriate amount of EDC and NHS solution was added to activate its carboxyl group, and after magnetic stirring for 30 min, 100 μL PEI solution was added to continue stirring for 2-5 h. After the above materials were washed several times, they were dispersed in 2 mL of ultrapure water, 100 μL of HAuCl ₄ ·6H ₂ O (1%) and 900 μL of NaBH ₄ (30 mM) were added and stirred overnight, and then dispersed in 2 mL of ultrapure water. The c-MWCNTs-PEI-AuNPs nanocomposite can be obtained in water, which is dispersed in 2 mL of ultrapure water and stored in a refrigerator at 4°C for later use;

Step 2. Polish the electrodes to a mirror surface with Al ₂ O ₃ powders of 0.3 μm and 0.05 μm respectively, and then use an appropriate amount of ultrapure water, absolute ethanol, and ultrapure water to sonicate the electrodes for 5 minutes each in the above order, and dry at room temperature for use;

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

Step 4. Drop 6 μL of sCD40L antibody on the surface of the modified electrode and incubate in a 4°C refrigerator for 10 hours;

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

Step 6. Rinse off the excess BSA on the surface of the electrode after incubation with ultrapure water, drop 6 μL of sCD40L with different concentrations on the electrode and incubate at 37°C for 45min;

Step 7. After the sCD40L that is not firmly bound to the sCD40L antibody was washed away with ultrapure water, it was placed in potassium ferricyanide solution (5mM K ₃ [Fe(CN) ₆ ], 5mM K ₄ [Fe(CN) ₆ ] ], 0.1M KCl) to characterize, and measure its current response value with DPV;

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

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

Step 10. The above-mentioned sensor in the present invention is stored in a refrigerator at 4°C, and the current response of the sensor is detected 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. The five electrochemical immunosensors of the present invention are used to detect sCD40L at the same concentration (1 pg mL ^-1 ), and the relative standard deviation is 1.38%, indicating that the sensor has good reproducibility;

Step 12. After using the present invention to detect sCD40L of the same concentration (1pg mL ^-1 ), put it into dissociation solution (30mM NaOH) for rinsing for 60s, take it out, carefully rinse it with ultrapure water and repeat the above steps, the results show that its Repeated 5 times the current value is still 95.79% of the initial current, indicating that the sensor has good reproducibility due to the stability of the material and the firm binding of the antibody and the material.

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.