A sensing system for specific detection of sialic acid, a preparation method and a sensor and a detection method

Abstract

A sensing system for specifically detecting sialic acid, a preparation method and a sensor and a detection method, using 4-mercapto phenylboronic acid as an analyte recognition molecule and using a PEG with a negative charge 400 The degree of intermolecular aggregation of gold nanoparticles is regulated after the modification of 4-mercapto phenylboronic acid, and high sensitivity and specific recognition of sialic acid molecules are realized. 525 / 660 The absorption intensity A of the prepared sensor and the sialic acid concentration are in a linear relationship R 2 =0.997, which has been successfully applied to the detection of sialic acid in biological serum and health care samples, and has shown application potential in the fields of clinical diagnosis and health care.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, specifically to a sensing system and its preparation method for the specific detection of sialic acid, as well as a sensor and detection method. Background Technology

[0002] N-acetylneuraminic acid (Neu5AcorNANA) is the most widely distributed sialic acid (SA) among carbohydrate α-keto acids, abundant in vertebrates and mammals. Sialic acid is a crucial component of glycoproteins, densely distributed on mammalian cell membranes, regulating key processes such as receiving external information, cell adhesion, and antigenicity. Studies have shown that sialic acid possesses diverse physiological functions, playing a vital role in regulating human physiological and pathological processes, such as promoting brain development, ion transport, membrane stabilization, and signal transduction. Furthermore, the expression level of sialic acid in serum provides important information for the diagnosis of malignant tumors, diabetes symptoms, and other related diseases. Previous studies have shown that exogenous sialic acid, when taken orally, can promote cell and tissue repair, cell division, and cell proliferation. Further research also indicates that SA, as a dietary supplement, can act as a cognitive enhancer. Therefore, an accurate and efficient method is needed to detect and quantify trace sialic acid levels in serum samples and dietary products for auxiliary diagnostic purposes and health supplement quality control.

[0003] Existing analytical methods such as colorimetric fluorescence, enzymatic methods, electrochemical biosensors, enhanced Raman scattering, quartz crystal microbalance (QCM), and chromatography are widely used for the detection of sialic acid. However, these methods all have limitations such as expensive equipment, complex analytical procedures, high requirements for operator expertise, and long processing times. Compared with other methods, colorimetric detection is simpler and has the advantage of visualizing test results.

[0004] Colloidal gold nanoparticles (AuNPs) are widely used in the design of colorimetric sensors due to their biofunctionalization, biostability, and spectral properties. By utilizing the plasmon resonance of gold nanoparticles and controlling their size and aggregation, changes in the color of colloidal solutions can be observed, providing a platform for the colorimetric detection of various analytes. According to the Derjaguin-Landua-Verwey-Overbeck (DLVO) theory, the stabilization of AuNPs can be controlled by the balance between the attractive van der Waals forces and the repulsive electrostatic double-layer forces between closely spaced adjacent particles. However, in complex assay systems, the aggregation state of gold nanoparticles can be caused by many uncontrollable factors, such as changes in salt concentration, electrostatic interference from charged biomolecules, or non-specific binding of biological components to unprotected active sites, which significantly affects the accuracy of measurements. Therefore, increasing research focuses on designing non-DLVO aggregation systems, i.e., modifying the surface of AuNPs with molecules that specifically capture analytes, to reduce assay interference caused by non-DLVO aggregation. However, various small molecules, polymers, or polyelectrolytes (e.g., gold-thiol, gold-amine) modified on the surface of gold nanoparticles also exhibit unpredictable cross-linking and aggregation. Therefore, how to regulate and reduce particle aggregation caused by intermolecular cross-linking has become an urgent task in designing a new generation of high-sensitivity colorimetric sensors. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a sensing system and preparation method for the specific detection of sialic acid, as well as a sensor and detection method that are simple to manufacture, highly specific, and have good test repeatability.

[0006] The technical solution adopted in this invention is: a sensing system for the specific detection of sialic acid, wherein the sensing system comprises 4-MPBA-AuNPs@PEG. 400 The nanocomposite material, namely 4-MPBA-AuNPs@PEG 400 Nanocomposite material is a polymer PEG used to stabilize gold nanoparticles. 400 Modified 4-MPBA-AuNPs, wherein the 4-MPBA-AuNPs are colloidal gold nanoparticles (AuNPs) with 4-mercaptophenylboronic acid (4-MPBA) attached to their surface for sialic acid recognition.

[0007] A method for preparing a sensing system for the specific detection of sialic acid includes the following steps:

[0008] (1) Preparation of colloidal gold solution: First, boil deionized water, then quickly add chloroauric acid solution and trisodium citrate solution, stir vigorously and continue to boil, then turn off the heating and continue stirring, cool to room temperature and then dilute to volume in a volumetric flask, store in a refrigerator for later use, and prepare colloidal gold solution.

[0009] (2) Preparation of 4-MPBA-AuNPs: Take the prepared colloidal gold solution into a clean container, adjust the pH to 11.0, add 4-MPBA solution dropwise, and incubate at room temperature to obtain 4-MPBA-AuNPs;

[0010] (3) 4-MPBA-AuNPs@PEG 400 Preparation: PEG was added dropwise to the prepared 4-MPBA-AuNPs. 400 After incubating the solution at room temperature, the mixture was transferred to a clean container and centrifuged. After centrifugation, the supernatant was removed, and the precipitate was redissolved in potassium hydroxide solution to obtain the 4-MPBA-AuNPs@PEG solution. 400 Solution.

[0011] In step (2), the amount of 4-MPBA solution added dropwise is 10µL of 0.05mol / L 4-MPBA solution.

[0012] In step (3), PEG is added dropwise. 400 The volume of solution is 0.25 mol / L of 1% PEG. 400 Solution.

[0013] In step (3), the centrifugation speed is 7500 rpm and the centrifugation time is 30 minutes.

[0014] The concentration of the potassium hydroxide solution in step (3) is 1×10⁻⁶. -11 mol / L.

[0015] A gold-based sensor based on a colorimetric sensing platform for the specific detection of sialic acid, the gold-based sensor comprising the aforementioned sensing system for the specific detection of sialic acid.

[0016] A method for specifically detecting sialic acid uses the aforementioned sensing system for specifically detecting sialic acid to detect sialic acid.

[0017] A specific method for detecting sialic acid involves using 4-MPBA-AuNPs@PEG. 400 The solution is dissolved in 0.01 mol / L buffer solution, and the sample solution to be tested is added. The solution color is observed or the intensity change of the absorption peak generated by ultraviolet spectroscopy is used for quantification.

[0018] Mechanism: The stability of the AuNP-based colorimetric sensing platform is designed by balancing DLVO-based and molecular-based interactions. 4-Mercaptophenylboronic acid (4-MPBA) linked to the AuNP surface acts as a receptor specifically recognizing sialic acid. The detection principle utilizes arylboronic compounds as ligands for affinity selection of sialic acid. To regulate the interparticle crosslinking and aggregation of functionalized gold nanoparticles, negatively charged polyethylene glycol 400 (PEG) is filled onto the unprotected gold surface. 400 This invention overcomes interparticle crosslinking by synergistically controlling parameters between two types of interactions, thus improving the sensor's testing performance. Simultaneously, biocompatible hydrophilic PEG... 400 With a strong hydration layer, it can prevent certain biomolecules from adsorbing or bacteria from attaching to the functionalized AuNP surface, which is beneficial for the detection of complex biological samples.

[0019] The beneficial effects of this invention are: This invention provides a sensing system and preparation method for the specific detection of sialic acid, as well as a sensor and detection method. By precisely controlling the balance between electrostatic attraction and molecular interactions, a sensing system and sensor for the specific detection of sialic acid are designed. The sensing system consists of two parts: 4-MPBA for sialic acid recognition and a polymer PEG. 400 4-MPBA-AuNPs@PEG were prepared to stabilize gold nanoparticles. 400 The sensor system is used for the highly selective and sensitive detection of sialic acid, with a detection limit of 48 μmol / L, providing a novel strategy to overcome the interparticle cross-linking and aggregation problems of existing colorimetric sensors. Furthermore, the proposed sensor system demonstrates great application potential in clinical diagnostics and health product quality monitoring. Attached Figure Description

[0020] Figure 1 (A) Transmission microscopy (TEM) of the prepared AuNPs and the particle size distribution shown in the inset; (B) UV absorption spectrum of the prepared AuNPs and a photograph of the sample.

[0021] Figure 2 shows a TEM image of 4-MPBA-AuNPs.

[0022] Figure 3 shows 4-MPBA-AuNPs@PEG. 400 TEM image.

[0023] Figure 4 shows AuNPs, 4-MPBA-AuNPs, and 4-MPBA-AuNPs@PEG. 400 The ultraviolet-visible absorption spectrum information.

[0024] Figure 5 shows 4-MPBA-AuNPs, 4-MPBA-AuNPs@PEG 400Raman spectral information.

[0025] Figure 6 4-MPBA-AuNPs@PEG 400 TEM image of the solution after adding sialic acid.

[0026] Figure 7 shows 4 MPBA-AuNPs@PEG 400 The ultraviolet spectral response of the sensor to different concentrations of sialic acid; where (A) color change; (B) intensity change of absorption peak; (C) calibration curve.

[0027] Figure 8 shows 4-MPBA-AuNPs@PEG. 400 Selectivity study of sialic acid by sensor testing Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] (1) Preparation of colloidal gold solution: First, boil 98 mL of deionized water, then quickly add 2 mL of 1% chloroauric acid solution and 8 mL of 1% trisodium citrate solution. Stir vigorously and continue boiling for 10 minutes. Then turn off the heating and continue stirring for 15 minutes. After cooling to room temperature, dilute to 100 mL in a volumetric flask and store at 4°C for later use. The morphology of the prepared colloidal gold was analyzed using high-resolution transmission electron microscopy, such as... Figure 1 As shown in Figure A, the prepared colloidal gold solution has a uniform particle size with an average particle size of 12.03 ± 0.80 nm; Figure 1 As shown in Figure B, a characteristic UV-Vis absorption peak for small-sized AuNPs was observed at 518 nm, which is consistent with... Figure 1 A. Information is consistent, insert Figure 1 B indicates that the prepared colloidal gold solution is wine-red in color and has good dispersibility.

[0030] (2) Preparation of 4-MPBA-AuNPs: Take 5 mL of the colloidal gold solution prepared in step (1) into a clean glass bottle, adjust the pH to 11.0 with KOH solution, and add 10 µL of 0.05 mol / L 4-MPBA solution dropwise. Incubate at room temperature for 12 hours. The morphology of the prepared 4-MPBA-AuNPs was analyzed by electron microscopy, as shown in the figure. Figure 2 As shown; the ultraviolet-visible absorption spectrum is as follows Figure 4 As shown; Raman spectroscopy analysis as follows Figure 5 As shown.

[0031] (3) 4-MPBA-AuNPs@PEG 400 Preparation: Add 0.25 mL of prepared 1% PEG dropwise to the 4-MPBA-AuNPs prepared in step (2). 400 After incubating the solution at room temperature for 30 minutes, transfer it to a clean centrifuge tube and centrifuge at 7500 rpm for 30 minutes. After centrifugation, remove the supernatant and reconstitute the precipitate to 1×10⁻⁶. -11 4-MPBA-AuNPs@PEG were obtained in mol / L potassium hydroxide solution. 400 The solution was stored at 4°C for later use. The prepared 4-MPBA-AuNPs@PEG 400 Morphological analysis was performed using electron microscopy, such as... Figure 3 As shown; the ultraviolet-visible absorption spectrum is as follows Figure 4 As shown; Figure 5 Raman spectroscopy clearly shows that 4-MPBA has been successfully modified onto the AuNP surface, and when small molecular weight PEG is added... 400 Subsequently, the characteristic peaks of 4-MPBA were preserved.

[0032] (4) Take the 4-MPBA-AuNPs@PEG obtained in step (3) 400 The solution was redissolved in 0.01 mol / L buffer (citric acid and potassium hydroxide buffer, pH=5.6). Then, sialic acid solutions of different concentrations were added, and 3 mL of each solution was transferred to a quartz cuvette. The 4-MPBA-AuNPs@PEG solution after the addition of sialic acid... 400 Solution morphology characterization by electron microscopy, such as Figure 6 As shown; under optimal test conditions, the 4 MPBA-AuNPs@PEG assay was validated. 400 The sensitivity of the sensing system to sialic acid detection. For example... Figure 7 As shown in Figure B, a new absorption peak is observed near 660 nm, indicating that 4-MPBA-AuNPs@PEG 400 When bound to sialic acid, the intensity of the absorption peak changes as the sialic acid concentration increases from 0.05 to 8 mmol / L, and the change in solution color with sialic acid concentration is visible to the naked eye. Figure 7 A) indicates that the system can achieve visual detection. 525 / A 660 The change in the ratio is linearly correlated with the concentration of sialic acid, such as... Figure 7 As shown in C. The calibration equation can be described as follows: A 525 / 600 = -0.17 C SA +2.14,R 2 =0.997 (concentration range 0.05-8 mM). Using this sensing system, the limit of detection (LOD) for sialic acid is estimated to be 48 μmol / L.

[0033] (5) Selectivity test: The 4-MPBA-AuNPs@PEG obtained in step (3) 400 Reconstituted into 0.01 mol / L buffer (citric acid and potassium hydroxide buffer, pH=5.6), and added different oligosaccharides of the same concentration (4 mmol / L), namely glucose (Glu), galactose (Gal), mannose (Man), sucrose (Suc), and sialic acid (SA) standards. The experimental results are as follows. Figure 8 As shown, other oligosaccharide compounds did not significantly interfere with the sialic acid test.

[0034] (6) Sample preparation: The sialic acid content in bird's nest was determined according to SN / T 3644-2016 "Determination of Sialic Acid in Exported Bird's Nest and its Products". 0.1g of ground bird's nest powder was dissolved in 10mL of 50% acetic acid solution and hydrolyzed in a water bath at 100℃ for 10 minutes. The supernatant was extracted and stored at 4℃. Sialic acid in serum was extracted by acidification. 5.56µL of sulfuric acid (18M) was mixed into 994.44µL of serum sample and hydrolyzed in a water bath at 80℃ for 60 minutes. Then, it was neutralized with 0.50mol / L KOH and kept for later use. (7) Sample detection: The standard addition method was used for testing. The test value was obtained by regression based on the calibration curve obtained in step (4). The results of the actual samples are shown in Table 1. The recovery rates of bird's nest and serum samples were 94.4-100.1% and 92.0-112.0%, respectively. Prove the reliability of the testing method and the feasibility of its practical application.

[0035] Table 1. Actual Samples Detected

[0036]

[0037] in conclusion

[0038] In summary, we designed and synthesized a novel gold-based sensor based on a colorimetric sensing platform by precisely controlling the balance between electrostatic attraction and molecular interactions. The sensor consists of two parts: 4-MPBA for sialic acid recognition and a polymer PEG... 400 Used to stabilize gold nanoparticles. The prepared 4-MPBA-AuNPs@PEG 400The sensing platform was developed for the highly selective and sensitive detection of SA, with a detection limit of 48 μmol / L. This study provides a novel strategy to overcome the interparticle cross-linking and aggregation issues inherent in existing colorimetric sensors. Furthermore, the proposed sensor system demonstrates significant application potential in clinical diagnostics and the quality control of health supplements.

[0039] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of protection of this patent.

[0040] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principle of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A sensing system for the specific detection of sialic acid, characterized in that, The sensing system includes 4-MPBA-AuNPs@PEG 400 The nanocomposite material, namely 4-MPBA-AuNPs@PEG 400 Nanocomposite materials are polymers such as PEG used to regulate the aggregation of gold nanoparticles. 400 The modified 4-MPBA-AuNPs, wherein the 4-MPBA-AuNPs are colloidal gold nanoparticles (AuNPs) with 4-mercaptophenylboronic acid (4-MPBA) attached to their surface for sialic acid recognition, are used to prepare the sensing system through the following steps: (1) Preparation of colloidal gold solution: First, boil deionized water, then quickly add chloroauric acid solution and trisodium citrate solution, stir vigorously and continue to boil, then turn off the heating and continue stirring, cool to room temperature and then dilute to volume in a volumetric flask, store in a refrigerator for later use, and prepare colloidal gold solution. (2) Preparation of 4-MPBA-AuNPs: Take the prepared colloidal gold solution into a clean container, adjust the pH to 11.0, add 10µL of 0.05mol / L 4-MPBA solution dropwise, and incubate at room temperature to obtain 4-MPBA-AuNPs; (3) 4-MPBA-AuNPs@PEG 400 Preparation: 0.25 mol / L 1% PEG was added dropwise to the prepared 4-MPBA-AuNPs. 400 After incubating the solution at room temperature, the mixture was transferred to a clean container and centrifuged. After centrifugation, the supernatant was removed, and the precipitate was redissolved in potassium hydroxide solution to obtain the 4-MPBA-AuNPs@PEG solution. 400 Solution.

2. A sensing system for the specific detection of sialic acid according to claim 1, characterized in that... In step (3), the centrifugation speed is 7500 rpm and the centrifugation time is 30 minutes.

3. A sensing system for the specific detection of sialic acid according to claim 1, characterized in that... In step (3), the concentration of the potassium hydroxide solution is 1×10⁻⁶. -11 mol / L.

4. A gold-based sensor based on a colorimetric sensing platform for the specific detection of sialic acid, characterized in that... The gold-based sensor comprises the sensing system for the specific detection of sialic acid as described in claim 1.

5. A method for specifically detecting sialic acid, characterized in that... Sialic acid is detected using the sensing system for specific detection of sialic acid as described in claim 1.

6. The method for specifically detecting sialic acid according to claim 5, characterized in that, 4-MPBA-AuNPs@PEG 400 The solution is dissolved in 0.01 mol / L buffer solution, and the sample solution to be tested is added. The solution color is observed or the intensity change of the absorption peak generated by ultraviolet spectroscopy is used for quantification.

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