CN107621492B

CN107621492B - Preparation method of biosensor for detecting alpha 2,3 sialylated glycan

Info

Publication number: CN107621492B
Application number: CN201710803305.XA
Authority: CN
Inventors: 于超; 何俊琳; 袁祺奇
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2017-09-06
Filing date: 2017-09-06
Publication date: 2020-09-11
Anticipated expiration: 2037-09-06
Also published as: CN107621492A

Abstract

α 2,3 sialylated glycan is one of tumor markers for early diagnosis of cancer, the work adopts a novel sandwich type biosensor based on 4-mercaptophenylboronic acid (4-MPBA), a novel molecular recognition system is constructed by matching boron atoms of 4-MPBA with amide groups of Neu5Ac and is used for detecting α 2,3 sialylated glycan for the first time, signal amplification is carried out by using an aminated fullerene palladium-platinum alloy, sophorea lectin is covalently immobilized on Au-polymethylene blue serving as a signal probe and is used for specifically recognizing α 2,3 sialylated glycan, and under the optimal experimental conditions, the biosensor shows 10fg mL^‑1～100ng mL^‑1Wide linear range of 3fg mL^‑1The detection limit of (S/N ═ 3) is low. In addition, the method shows good repeatability and stability, indicating that it can be used in clinical studies.

Description

Preparation method of biosensor for detecting

alpha

2,3 sialylated glycan

The technical field is as follows:

the invention relates to a preparation method and application of an electrochemical sensor for clinically and quantitatively diagnosing detection of

alpha

2,3 sialylated glycans, in particular to a sandwich type biosensor prepared based on auromethylidene blue-sophorae Huai agglutinin as a nano beacon, which is used for detecting the

alpha

2,3 sialylated glycans and belongs to the field of electrochemical detection.

Background art:

sialic acid is attached to cell surface glycoconjugates as a terminal monosaccharide due to its nine-carbon structure. The

α

2,3 linkage of sialic acid to N-acetyllactosamine structure (Gal β 1-4GlcNAc) is a Golgi mediated process mediated by β -

galactoside α

2,3 sialyltransferase (ST3 Gal-I). Variants of

α

2,3 sialylation are associated with a wide range of biological and pathological diseases, including the development of certain cancers. When tumor tissue undergoes apoptosis, α 2, 3-sialylglycan (α 2, 3-sial-Gs) is synthesized and released into the blood, resulting in an elevated level of

α

2,3 sialylation in the patient's blood. Thus, high levels of

α

2,3 sialylation can be detected in the blood of some cancer patients. Early detection, disease monitoring and accurate prediction have important applications in clinical analysis of biomarker molecules in human serum samples. Therefore, in recent years, great efforts have been made to achieve highly

sensitive α

2,3 sialylation detection.

To date, only a few analytical methods have been available for the detection of polysaccharides in saliva, including electrospray Mass Spectrometry (MS), tandem MS and miniaturized glycosyltransferase assays. Although these methods have some advantages, such as good qualitative ability, classical and high resolution techniques. However, they suffer from the common disadvantages of long analysis times, high precision instruments and high technician requirements, which limit their widespread use. Therefore, attempts to explore and study new detection techniques for early and sensitive analysis of cancer biomarkers are of great importance, especially in clinical applications. In recent years, sandwich biosensors have been one of the important analytical techniques for sensitive and specific detection of small biomolecules and proteins, and have been widely used in biomedical research.

Neu5Ac a (2-3) Gal β MP glycoside, a recombinant soluble form of a 2,3 sialylated glycan, formed by the a 2,3 linkage of sialic acid with the N-acetyl lactose structure in glycoconjugates, which can be recognized by specific lectins. Certain sia-binding lectins have been demonstrated to be effective tools for the detection of specific glycoconjugates. The locust lectin (MAL) is an ideal tool for specifically detecting alpha 2, 3-meal-Gs, and can be specifically combined with the alpha 2, 3-meal-Gs. However, due to the specific structure of α 2, 3-meal-Gs, one MAL molecule can be combined with one α 2, 3-meal-Gs, making sensitive detection difficult. In order to sensitively detect alpha 2, 3-sial-Gs, 4-mercaptophenylboronic acid (4-MPBA) was proposed for the first time as a capture molecule. Formation of a triangular compound stabilized by coordinating the boron atom of 4-MPBA with the amide group of Neu5Ac forms an intramolecular B-O bond. Thus, a sensitive and specific sandwich-type biosensor based on 4-MPBA has been constructed, which is used as a novel recognition molecule for detecting α 2, 3-sial-Gs in human serum.

Methylene Blue (MB) is a derivative of a phenothiazine dye, useful for electrochemical redox active substances and signal materials in biosensors. However, when MB is used alone, it is difficult to generate a stable signal, which limits its applications. Therefore, the research adopts Au nanoparticles, and the stability of MB is improved due to high conductivity, large specific surface area, good biocompatibility and strong absorption of biomolecules. MAL functionalized poly (methylene blue) (MAL-Au-PMB) is conveniently synthesized by oxidative polymerization of MB and serves as a novel redox species for the first time for α 2, 3-sil-Gs detection.

In recent years, metal alloys have attracted considerable attention because of their excellent physicochemical properties. Due to the high conductivity, good stability, and increased surface area, alloys of palladium and platinum (PdPt) are used. For the capacity of loading PdPt, amino-functionalized fullerene (n-C) with large surface area is introduced₆₀) Excellent electron acceptor capability and abundant amino groups for further modification. Thus, due to PdPt alloy and n-C₆₀The advantages of the two are that n-C with large loading capacity and good conductivity is synthesized through the stable connection between the noble metal nano particles and the amino₆₀The work provides a sandwich type biosensor based on 4-MPBA, and the sandwich type biosensor can be applied to high-sensitivity detection of α 2, 3-sial-Gs in human serum for the first time.

In this study, 4-MPBA was found and used to detect α 2, 3-sial-Gs, resulting in a sandwich-type electrochemical biosensor, which was used for the first time to detectα 2, 3-sial-Gs structure to realize sensitive detection, n-C is adopted₆₀PdPt and Au-PMB composite material. Using n-C with large surface area and good conductivity₆₀PdPt to immobilize more 4-MPBA. Au-PMB capable of capturing α, 3-sial-Gs as a signal probe for amplifying electrochemical signals can immobilize MAL through Au-NH2 bond, and then Au-PMB-MAL specifically combines with α, 3-sial-Gs and generates electrochemical signals.

The invention content is as follows:

1. the invention aims to provide a preparation method and application of a sandwich type electrochemical biosensor for detecting

alpha

2,3 sialylated glycan, which is characterized by comprising the following steps:

(1) preparing an aminated fullerene (n-C60) -palladium-platinum (Pdpt) complex;

(2) preparing gold (Au) -Methylene Blue (MB) -sophorae lectin (MAL) nano-beacons;

(3) and (3) establishing a sandwich type electrochemical biosensor, measuring

alpha

2 and 3 sialylated glycan, and drawing a standard curve.

2. The preparation process of the n-C60-PdPt compound and the Au-PMB-MAL nano beacon is characterized by comprising the following steps of:

(1)n-C₆₀preparation of PdPt nanocomposites:

first, 2mg of n-C₆₀Dispersed in 1mL of 0.1M HCl and then sonicated for 2 hours until n-C₆₀Uniformly dissolved in the solution. Next, 2mL of ultrapure water was added to the solution. The mixture was then stirred and heated to 100 ℃. Thereafter, 150mg of AA, 1.17mg of K2 PtCl 4 and 1.66mg of Na 2PdCl4 were added to 1mL of ultrapure water, respectively. After heating at 100 ℃ for 2.5 hours, the product was cooled to room temperature. Then, the mixture was mixed at 8000rpm min^-1Centrifuge for 3 minutes and wash three times to remove remaining AA, free Pt and Pd nanoparticles. Finally, the precipitate was dissolved in 1mL of ultrapure water before use for the next step

(2) Preparing an Au-PMB nano material:

first, MB (9.8mM, 1mL) was added to a solution of HCl (0.1M, 600. mu.L) and DTAB (1.14mM, 6mL) and stirred well for about 10 minutes. Subsequently, HAuCl 4 (4%, 200 μ L) was added to the compound and stirring was continued at room temperature for about 6 hours. The compound was centrifuged at 8000rpm min-1 for 5 minutes and washed three times with ultrapure water. Finally, the compound was dispersed in 2mL of ultrapure water.

(3) Preparing an Au-PMB-MAL nano beacon:

first, 1mLAu-PMB complex and 10. mu.LMAL (2mg mL)^-1) Mix gently and stir at 4 ℃ for 12 hours. Subsequently, the mixture was run at 8000rpm min^-1Centrifuge for 5 minutes and wash three times to remove unbound MAL. And the precipitate was dissolved in 1mL of ultrapure water. Next, 500. mu.L of 1 w% BSA was added to the compound and gently mixed at 4 ℃ for 1 hour, and then the mixture was mixed at 8000rpm for min^-1Centrifuge for 5 minutes and wash thoroughly 3 times to eliminate excess BSA. Finally, the compound was dispersed in 2mL of ultrapure water.

3. The method for establishing a sandwich-type electrochemical biosensor according to claim 1, measuring

α

2,3 sialylated glycans, plotting a standard curve, characterized by comprising the steps of:

(1) with 0.3 and 0.05 μm Al, respectively₂O₃Polishing the electrode into a mirror surface by using powder, then respectively carrying out ultrasonic treatment on the electrode for 5min according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying at room temperature for later use;

(2) 8 mu.L of prepared n-C₆₀Dropwise adding PdPt electrode modification material on the surface of the electrode, and drying at room temperature;

(3) 10 mu.L of 1.0m mol-14-MPBA solution is dripped into n-C₆₀PdPt/GCE and incubation at 4 ℃ for 12 hours;

(4) after thorough washing with ultrapure water, 10. mu.L of 1 w% BSA was dropped on the electrode surface for 30 minutes to prevent non-specific binding sites;

(5) after washing with water, standard α 2, 3-sil-Gs solutions of different concentrations were incubated at 4 ℃ for 2 hours on the electrode surface.

(6) mu.L of the pretreated Au-PMB-MAL complex was dropped onto the modified electrode and incubated at 4 ℃ for 2 hours.

(7) And drawing a working curve according to the linear relation between the obtained current change value and the concentration of the standard alpha 2, 3-sial-Gs solution.

Compared with the prior art, the preparation method and the application of the sandwich type electrochemical biosensor for detecting the

alpha

2,3 sialylated glycan have the outstanding characteristics that:

(1) will be based on n-C₆₀The nano composite material of PdPt is introduced into the preparation of the electrochemical biosensor as an electrode modification material, so that the immobilization amount of biomolecules is effectively improved, the conductivity is increased, and the sensitivity of the electrochemical biosensor is improved;

(2) the boron atom of the 4-MPBA is coordinated with the amido of the Neu5Ac to form a stable triangular compound, an intramolecular B-O bond is formed, more alpha 2, 3-sial-Gs can be captured, and the sensitivity of the sensor is improved;

(3) by adopting the MAL-Au-PMB as the nano beacon of the research, the nano beacon not only has the advantages of high conductivity, large specific surface area and good biocompatibility, but also improves the stability of MB and generates stable electric signals;

(4) the prepared biosensor shows good performance on the analysis of alpha 2, 3-sial-Gs, and has the potential of being applied to the detection of other sialylated glycans. In addition, the method is sensitive, has a wide application range, and is convenient to realize commercialization, thereby promoting the development of precise medicine.

Description of the drawings:

FIG. 1 is a schematic view showing the construction of a sandwich-type electrochemical biosensor according to the present invention.

FIG. 2 is a scanning electron micrograph, a transmission electron micrograph, an EDS chart and an XPS chart of different synthetic steps of the nanocomposite material of the present invention.

FIG. 3 is a UV, IR, EDS and Zeta potential diagram of the nanocomposites of the present invention at different synthesis steps.

FIG. 4 is an atomic force microscope image of the nanocomposite of the invention at different synthetic steps.

FIG. 5 is a graph showing the linear relationship between the current and the concentration obtained when the electrochemical sandwich type biosensor of the present invention detects α 2, 3-sial-Gs, and the impedance of the sensor.

The specific implementation mode is as follows:

the invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.

Example 1

Step 1. first, 2mg of n-C60 was dispersed in 1mL of 0.1M HCl and then sonicated for 2 hours until n-C60 was uniformly dissolved in the solution. Next, 2mL of ultrapure water was added to the solution. The mixture was then stirred and heated to 100 ℃. Thereafter, 150mg of AA, 1.17mg of K2 PtCl 4 and 1.66mg of Na 2PdCl4 were added to 1mL of ultrapure water, respectively. After heating at 100 ℃ for 2.5 hours, the product was cooled to room temperature. Then, the mixture was mixed at 8000rpm min^-1Centrifuge for 3 minutes and wash three times to remove remaining AA, free Pt and Pd nanoparticles. Finally, the precipitate was dissolved in 1mL of ultrapure water before use for the next step.

Step 2. first, MB (9.8mM, 1mL) was added to a solution of HCl (0.1M, 600. mu.L) and DTAB (1.14mM, 6mL) and stirred well for about 10 minutes. Subsequently, HAuCl 4 (4%, 200 μ L) was added to the compound and stirring was continued at room temperature for about 6 hours. The compound was run at 8000rpm min^-1Centrifuged for 5 minutes and washed three times with ultrapure water. Finally, the compound was dispersed in 2mL of ultrapure water.

Step 3. first, 1mL of Au-PMB complex and 10. mu.L of MAL (2mg mL-1) were gently mixed and stirred at 4 ℃ for 12 hours. Subsequently, the mixture was run at 8000rpm min^-1Centrifuge for 5 minutes and wash three times to remove unbound MAL. And the precipitate was dissolved in 1mL of ultrapure water. Next, 500. mu.L of 1 w% BSA was added to the compound and gently mixed at 4 ℃ for 1 hour, and then the mixture was mixed at 8000rpm for min^-1Centrifuge for 5 minutes and wash thoroughly 3 times to eliminate excess BSA. Finally, the compound was dispersed in 2mL of ultrapure water.

Step 4. use of 0.3 and 0.05 μm, respectivelyAl₂O₃Polishing the electrode into a mirror surface by using powder, then respectively carrying out ultrasonic treatment on the electrode for 5min according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying at room temperature for later use;

step 5. preparing n-C with 8 μ L₆₀Dropwise adding PdPt electrode modification material on the surface of the electrode, and drying at room temperature;

step 6, dripping 10 mu L of 1.0m mol-14-MPBA solution into n-C₆₀PdPt/GCE and incubation at 4 ℃ for 12 hours;

step 7, washing the incubated electrode with ultrapure water, then dropwise adding 10 mu L of DNA capture probe solution marked by 1 mu M of biotin, and incubating for 12h at 4 ℃;

step 8, after thorough washing with ultrapure water, 10 microliter of 1 w% BSA is dripped on the surface of the electrode for 30 minutes to prevent non-specific binding sites;

step 9. after washing with water, standard α 2, 3-sil-Gs solutions of different concentrations were incubated at 4 ℃ for 2 hours on the electrode surface.

Step 10, dripping 10 mu L of the pretreated Au-PMB-MAL compound on the modified electrode, and incubating for 2 hours at 4 ℃;

step 11, drawing a working curve according to the linear relation between the obtained current change value and the concentration of the standard α 2, 3-sial-Gs solution, wherein the measurement result shows that the concentration of the standard α 2, 3-sial-Gs solution is 10fg mL^-1～100ng mL^-1Linear relation in the range, linear correlation coefficient of 0.9971, detection limit of 0.3fg mL^-1。

Step 12, storing the sensor at 4 ℃, detecting the current response of the sensor, wherein the current response is still 89.63 percent of the initial current after 28 days of storage, which indicates that the sensor has good stability;

step 13, taking 5 sandwich type biosensors prepared in the same batch, and carrying out the detection on 5pgmL of the biosensors under the same condition^-1α 2, 3-sial-Gs solution is measured respectively, each electrode is measured for 3 times, the relative standard deviation of response current is 1.52%, and the result verifies that the sandwich type sensor has good reproducibility.

And step 17, using the sensor of the invention to detect target sialylated glycan, alpha 2, 6-sial-Gs, ascorbic acid, dopamine, glucose, L-cysteine and bovine serum albumin. These experiments demonstrated that the change in current only comes from the specific recognition between α 2, 3-sil-Gs and lectin. The change in current caused by the interfering substances was less than without these compounds, indicating that the selectivity of the sandwich biosensor was acceptable.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a biosensor for detecting α 2,3 sialylated glycans, comprising the steps of:

(1) amination of C₆₀Preparation of palladium platinum nanocomposites:

first, 2mg of aminated C₆₀Dispersed in 1mL, 0.1M hydrochloric acid and then sonicated for 2 hours until amination C₆₀Uniformly dissolving in the solution, next, adding 2mL of ultrapure water to the above solution, followed by stirring and heating to 100 ℃, after that, adding 150mg of ascorbic acid, 1.17mg of potassium tetrachloroplatinate and 1.66mg of sodium tetrachloropalladate to 1mL of ultrapure water, and after heating at 100 ℃ for 2.5 hours, cooling the product to room temperature, then, centrifuging the reacted solution at 8000rpm/min for 3 minutes and washing three times to remove the remaining ascorbic acid, free platinum and palladium nanoparticles, and finally, dissolving the precipitate in 1mL of ultrapure water for the next step before use;

(2) preparing a gold-polymethylene blue nano material:

first, 1mL of 9.8mM methylene blue was added to 0.1M, 600. mu.L of hydrochloric acid and 1.14mM, 6mL of dodecyltrimethylammonium bromide solution, and stirred well for about 10 minutes, then 200. mu.L of 4% chloroauric acid solution was added to the above solution and stirred continuously at room temperature for about 6 hours, and the reaction-completed solution was stirred at 8000rpm for min^-1The mixture is centrifuged for 5 minutes and then,washing with ultrapure water for three times, and finally, re-dispersing the precipitate in 2mL of ultrapure water;

(3) preparing a gold-polymethylene blue-sophorae Huai agglutinin nano beacon:

first, 1mL of the prepared gold-polymethylene blue solution and 10. mu.L, 2mg mL of the prepared gold-polymethylene blue solution were mixed^-1The sophorae lectin solution was gently mixed and stirred at 4 ℃ for 12 hours, and then, the mixture was stirred at 8000rpm for min^-1Centrifuging for 5 minutes and washing three times to remove unbound Sophora japonica lectin, and dissolving the precipitate in 1mL of ultrapure water, then, adding 500. mu.L, 1 w% bovine serum albumin to the above solution and gently mixing at 4 ℃ for 1 hour, and then mixing the mixed solution at 8000rpmmin^-1Centrifugation for 5 minutes, thorough washing 3 times to eliminate excess BSA, and finally, dispersion of the collected precipitate in 2mL of ultrapure water;

(4) establishing a sandwich type electrochemical biosensor:

a. polishing the electrodes into mirror surfaces by using 0.3 and 0.05 mu m aluminum oxide powder respectively, then carrying out ultrasonic treatment on the electrodes for 5min respectively according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying at room temperature for later use;

b. 8 μ L of prepared aminated C₆₀Dripping the palladium-platinum nano composite material on the surface of the GCE, and drying at room temperature;

c. mixing 10 μ L of 1.0m mol^-14-mercaptophenylboronic acid solution is dripped into amination C₆₀-palladium platinum modified GCE and incubation at 4 ℃ for 12 hours;

d. after thorough washing with ultrapure water, 10. mu.L of 1 w% BSA was added dropwise to the electrode surface for 30 minutes to prevent non-specific binding sites.

2. The method for the quantitative detection of α 2,3 sialylated glycans achieved by the sensor obtained by the preparation method according to claim 1, characterized by comprising the steps of:

(1) dripping alpha 2,3 sialylated glycan solutions with different concentrations on the surface of the constructed sensor, and incubating for 2 hours at 4 ℃;

(2) dripping 10 μ L of pretreated gold-polymethylene blue-sophorae lectin nano beacon on the modified electrode, and incubating at 4 deg.C for 2 hr;

(3) and drawing a working curve according to the linear relation between the obtained current change value and the concentration of the alpha 2,3 sialylated glycan solution.