FRET-based fluorescent probe, preparation method and application in detecting sialic acid

Abstract

The present invention provides a FRET-based fluorescent probe comprising the structure shown in Formula 1: R is selected from one of H, a sulfonic acid group, and a sulfonate salt. The present invention also provides a method for preparing the fluorescent probe and its use in detecting sialic acid. This fluorescent probe is simple to prepare and exhibits excellent single-excitation dual-emission performance. Used as a probe, it enables fluorescence ratiometric detection of sialic acid, avoiding the vulnerability of single-wavelength fluorescence detection to probe concentration, environmental factors, and other factors. The detection limit reaches 1.83 μM.

Description

Technical Field

[0001] The patent of this invention relates to the field of analysis and detection technology, and in particular refers to a fluorescent probe based on FRET (fluorescence resonance energy transfer), its preparation method and its application in detecting sialic acid. Background Art

[0002] Sialic acid, a neuraminic acid derivative with multiple biological functions, including antiviral, antioxidant, and immunomodulatory properties, is currently used in a wide range of products, including edible bird's nests, milk powder, and pharmaceuticals. Furthermore, high concentrations of sialic acid in body fluids are closely associated with the development of cancer and serve as a key tumor marker. Therefore, establishing a rapid and accurate sialic acid analysis method is crucial for evaluating the quality of related products and determining the occurrence of cancer.

[0003] Over the past few decades, researchers have developed a variety of sialic acid detection and analysis methods, such as colorimetry, electrochemical methods, high-performance liquid chromatography, liquid chromatography-mass spectrometry, and fluorescence analysis. Among them, fluorescence analysis has attracted increasing attention from researchers in recent years due to its high sensitivity, good selectivity, ease of operation, and low cost. A variety of fluorescent materials have been developed for the detection of sialic acid. For example, Yu et al. (Talanta 2021; 232: 122434) synthesized a boronic acid-modified metal-organic framework UiO-66-NH2@B(OH)2 by a one-step solvothermal method for fluorescence quenching detection of sialic acid; Wang et al. (New Journal of Chemistry 2020; 44: 2350-2356) reported a near-infrared carbon dot synthesized by a one-pot method using o-phenylenediamine and 3-aminophenylboronic acid as precursors for fluorescence enhanced detection of sialic acid. Wang et al. (RSC Advances 2016; 6:481) prepared a magnetic fluorescent nanoparticle for detecting sialic acid in infant formula. Currently reported fluorescent probes have certain advantages, but still have some shortcomings, such as complex and time-consuming synthesis and are mainly used for single-wavelength detection.

[0004] Ratiometric fluorescence detection is based on the ratio of fluorescence signals at two different wavelengths to achieve analytical determination. Due to its self-correction capability, it can effectively avoid interference from environmental factors and improve the specificity and accuracy of detection. However, there are currently few studies on ratiometric fluorescence probes for detecting sialic acid. Summary of the Invention

[0005] FRET refers to the phenomenon in which two chromophores (donor and acceptor molecules) come into close proximity. After one donor molecule absorbs energy, it is transferred to an adjacent acceptor molecule through resonance, causing it to become excited and emit fluorescence. As a fluorescence ratiometric detection mechanism, FRET technology has been widely used in the analysis and detection of small molecules, ions, and macromolecules.

[0006] In this study, we used quinolineboronic acid and a 1,2-dihydroxyanthraquinone derivative (alizarin red or alizarin) as raw materials to synthesize an in situ fluorescent probe. Upon excitation, this probe undergoes intramolecular FRET from quinoline to anthraquinone, exhibiting single excitation and double emission characteristics. This probe can be directly used for fluorescence ratiometric detection of sialic acid without the need for isolation or purification, providing a new approach and method for the determination of sialic acid.

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. Therefore, in a first aspect of the present invention, the present invention provides a FRET-based fluorescent probe, wherein the FRET-based fluorescent probe comprises a structure shown in Formula 1:

[0008]

[0009] R is selected from one of H, sulfonic acid group and sulfonate.

[0010] In one or more embodiments of the present invention, the FRET-based fluorescent probe comprises a structure shown in the formula QTB-ARS:

[0011]

[0012] In a second aspect of the present invention, the present invention provides a method for preparing the FRET-based fluorescent probe described in the first aspect of the present invention, comprising: mixing quinolineboronic acid and a 1,2-dihydroxyanthraquinone derivative to obtain the FRET-based fluorescent probe.

[0013] In one or more embodiments of the present invention, the quinoline boronic acid structure is: The structure of the 1,2-dihydroxyanthraquinone derivative is:

[0014] Preferably, the quinolineboric acid is 3-quinolineboric acid; and the 1,2-dihydroxyanthraquinone derivative is alizarin red or alizarin.

[0015] In one or more embodiments of the present invention, the mixing is performed in a solvent, and the solvent is selected from at least one of ethanol, methanol, tetrahydrofuran, acetone, water and acetonitrile.

[0016] In the third aspect of the present invention, the present invention provides a kit comprising the FRET-based fluorescent probe described in the first aspect of the present invention or the FRET-based fluorescent probe prepared by the preparation method described in the second aspect of the present invention.

[0017] In one or more embodiments of the present invention, the kit further comprises a buffer solution and cetyltrimethylammonium bromide (CTAB) at a concentration of 2.0-15.0 mM, and the pH of the buffer solution is 4.0-6.0.

[0018] The buffer solution is selected from at least one of phosphate buffer solution, acetate-sodium acetate buffer solution, acetate-ammonium acetate buffer solution and acetate-potassium acetate buffer solution.

[0019] In the fourth aspect of the present invention, the present invention provides a use of the FRET-based fluorescent probe described in the first aspect of the present invention, the FRET-based fluorescent probe prepared by the preparation method described in the second aspect of the present invention, or the kit described in the third aspect of the present invention in detecting sialic acid.

[0020] In a fifth aspect of the present invention, the present invention provides a method for detecting sialic acid, characterized in that it comprises the following steps:

[0021] 1) The FRET-based fluorescent probe described in the first aspect of the present invention or the FRET-based fluorescent probe prepared by the preparation method described in the second aspect of the present invention is added to a buffer solution, and then a CTAB solution is added to the constant volume to obtain a detection system. The fluorescence intensity at wavelengths of 400 nm and 580 nm is measured respectively, and the ratio of the fluorescence intensity at wavelengths of 400 nm to 580 nm is calculated. 400 / 580 ;

[0022] 2) Add the test sample containing sialic acid to the above detection system, measure the fluorescence intensity at 400nm and 580nm wavelengths respectively, and calculate the ratio of the fluorescence intensity at 400nm to 580nm wavelengths F1 400 / 580 , through F1 400 / 580 / F0 400 / 580 The linear relationship between the sialic acid concentration and the sialic acid concentration is achieved, thereby realizing the quantitative measurement of the sialic acid test sample.

[0023] In one or more embodiments of the present invention, in step 1), the volume ratio of the FRET-based fluorescent probe to the detection system is 15-25 μL:1 mL.

[0024] The present invention has the following advantages:

[0025] The present invention provides a FRET-based fluorescent probe. This probe is simple to prepare and exhibits excellent single-excitation dual-emission performance. It can be used as a probe for fluorescence ratiometric detection of sialic acid, avoiding the vulnerability of single-wavelength fluorescence detection to probe concentration and environmental factors. The detection limit for sialic acid is 1.83 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1The fluorescence spectra of the mixed solutions of 3-quinolineboronic acid (QTB) and alizarin red (ARS) at different concentrations in Example 1 (QTB concentration: 100 μM; ARS concentration: 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM; λ ex =320nm);

[0027] Figure 2 UV spectra of the alizarin red (ARS) solution, 3-quinolineboronic acid (QTB) solution, and the mixed solution of alizarin red and 3-quinolineboronic acid (QTB-ARS) in Example 2;

[0028] Figure 3 The structure and mass spectrum of QTB-ARS in the FRET-based fluorescent probe prepared in Example 3;

[0029] Figure 4 This is the fluorescence response emission spectrum of the FRET fluorescent probe prepared in Example 4 to sialic acid (excitation wavelength 320 nm);

[0030] Figure 5 The fluorescence signal F1 in the fluorescence ratio determination of sialic acid by the FRET fluorescent probe prepared in Example 4 is 400 / 580 / F0 400 / 580 The fitted straight line between the sialic acid concentration and the DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with specific examples and accompanying drawings, but the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. In the following examples, if no specific conditions are specified, the conditions according to normal conditions or manufacturer's recommendations are carried out, and the method used, if not otherwise specified, is a conventional method well known in the art, and the consumables and reagents used, if not otherwise specified, are commercially available. Unless otherwise indicated, the professional and scientific terms used herein are identical in meaning to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied to the present invention.

[0032] Example 1: Energy transfer between 3-quinolineboronic acid and Alizarin Red

[0033] Prepare the following concentrations of 3-quinolineboronic acid and alizarin red mixed solutions: keep the QTB (3-quinolineboronic acid) concentration at 100μM (solvent is ethanol), and the ARS (alizarin red) concentrations are 20μM, 40μM, 60μM, 80μM, 100μM, 110μM, 120μM, 130μM, 140μM, 150μM (solvent is water). After the solution is mixed and allowed to stand for 20 minutes, the fluorescence emission spectra of the 3-quinolineboronic acid and alizarin red mixed solutions at different alizarin red concentrations are measured with an excitation wavelength of 320nm. Figure 1 As the concentration of Alizarin Red increases, the fluorescence intensity at 580 nm of the mixed solution gradually increases. At 100 μM, the fluorescence intensity at 580 nm reaches its maximum. Further increases in Alizarin Red concentration result in little change in the fluorescence intensities at 400 nm and 580 nm. This experiment demonstrates that upon mixing 3-quinolinylboronic acid and Alizarin Red, FRET occurs between the quinoline fluorophore and the anthraquinone fluorophore.

[0034] Example 2: UV Spectrometry of Alizarin Red, 3-Quinolineboric Acid, and a Mixed Solution of Alizarin Red and 3-Quinolineboric Acid

[0035] The UV-Vis absorption spectra of quinoline-3-boronic acid solution (50 μM) (solvent: ethanol), alizarin red solution (50 μM) (solvent: water), and a mixed solution of quinoline-3-boronic acid solution (100 μM) (solvent: ethanol) and alizarin red solution (100 μM) (solvent: water) in equal volumes were measured. The results are as follows: Figure 2 As shown in the figure, 3-quinolineboronic acid has an absorption peak at 315nm and no absorption peak above 400nm; while Alizarin Red has distinct absorption peaks at 260nm, 340nm, and 530nm. The UV spectrum of a mixed solution of the two shows no absorption peak at 530nm, a characteristic absorption peak at 460nm, and strong absorption at 260nm and 315nm. This change confirms the formation of the new compound QTB-ARS.

[0036] Example 3: Mass spectrometry test of QTB-ARS

[0037] Take equal volumes of 3-quinolinylboronic acid (100 μM) (solvent: ethanol) and Alizarin Red (100 μM) (solvent: water) solution and mix them to obtain a FRET-based fluorescent probe. The MS spectrum was measured by electrospray ionization in the positive ion detection mode. The results are as follows: Figure 3 As shown. The literature reports that boric acid compounds can covalently bind to substances with vicinal diol structures to form a pentavalent borate structure. Therefore, the inventors speculated that 3-quinolineboronic acid and alizarin red reacted to form the product QTB-ARS, the molecular formula of which is C 23 H 11BNO7S. The structural formula of the product QTB-ARS is shown below:

[0038]

[0039] from Figure 3 The mass spectrum analysis results show that there is an ion fragment with a mass-to-charge ratio of m / z = 473.92, corresponding to [QTB-ARS+NH4] + , which is consistent with the theoretical calculated value of 474.06, proving the generation of QTB-ARS.

[0040] Example 4: QTB-ARS fluorescent probe for fluorescence ratiometric determination of sialic acid

[0041] A 5mM quinoline-3-boronic acid (ethanol) and a 5mM alizarin red (water) stock solution were mixed in a 1:1 ratio and allowed to stand for 20 minutes to obtain a probe solution containing QTB-ARS. 20 μL of the QTB-ARS probe solution was added to a test tube, followed by 0.5 mL of acetic acid-sodium acetate buffer (20 mM, pH = 5.5), followed by 200 μL of 50 mM CTAB solution, and finally, sialic acid solutions of varying concentrations were added. The volume was then adjusted to 1 mL with first-grade ultrapure water, and the mixture was thoroughly shaken and the fluorescence spectrum was measured at an excitation wavelength of 320 nm. The results are shown in FIG. Figure 4 As shown. Record the fluorescence intensity of the detection system at 400nm and 580nm under different sialic acid concentrations. Figure 4 As shown in the figure, within the sialic acid concentration range of 0-10 mM, as the sialic acid concentration gradually increases, the fluorescence intensity of the detection system at 400 nm gradually increases, and the fluorescence intensity at 580 nm gradually decreases. 400 / 580 Gradually increase. 400 / 580 / F0 400 / 580 (F0 400 / 580 and F1 400 / 580 The ratio of the fluorescence intensity at 400 nm to that at 580 nm before and after the addition of sialic acid is taken as the ordinate, and the sialic acid concentration is taken as the abscissa. The linear fitting results are as follows: Figure 5 The linear range of sialic acid determination is 0-10 mM, and the detection limit of sialic acid determination is calculated to be 1.83 μM based on the signal-to-noise ratio (S / N) of 3.

[0042] Example 5: QTB-ARS fluorescent probe for detecting sialic acid in milk powder

[0043] Weigh 3.0g of commercially available milk powder accurately, place it in a 50mL volumetric flask, and dilute to the mark with ultrapure water. Dissolve the solution, then transfer 900μL to a 10mL EP tube. Accurately add 5mL of 0.05M sulfuric acid solution. Hydrolyze the solution in an 85°C water bath for 1 hour, remove the solution, cool it to room temperature, and adjust it to neutral with 1M NaOH solution. Dose a certain amount of the solution and centrifuge it to obtain the supernatant, which is the sample solution. Add a certain amount of the sample solution to the quantitative sialic acid standard solution to obtain the spiked sample solution.

[0044] Take 20 μL of the probe solution containing QTB-ARS prepared in Example 4 and add it to a test tube. Then add 0.5 mL of acetic acid-sodium acetate buffer (20 mM, pH = 5.5), then add 200 μL of CTAB solution (50 mM), and finally add a certain amount of milk powder sample solution or spiked sample solution. The volume is made up to 1 mL with ultrapure water. After thorough shaking and mixing, the fluorescence spectrum is measured at an excitation wavelength of 320 nm, and the fluorescence intensity ratio F1 is calculated. 400 / 580 At the same time, the fluorescence spectrum of the blank control without adding sample solution was measured and the fluorescence intensity ratio F0 was calculated. 400 / 580 By fluorescence intensity ratio F1 400 / 580 / F0 400 / 580 The linear relationship between the QTB-ARS probe and sialic acid was established, and the spiked recovery of sialic acid in the test samples was calculated. The spiked recovery of sialic acid in actual milk powder samples ranged from 101.2% to 104.6%, with a relative standard deviation of less than 4%, demonstrating the feasibility of the QTB-ARS probe in detecting sialic acid in real samples.

[0045] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention, and all of these changes shall be included within the scope of protection of the present invention.

Claims

1. A FRET-based fluorescent probe, characterized in that: The FRET-based fluorescent probe is selected from the structure shown in Formula 1: R is selected from one of H, sulfonic acid group and sulfonate.

2. The FRET-based fluorescent probe according to claim 1, wherein The FRET-based fluorescent probe is selected from the structure shown in the formula QTB-ARS:

3. A method for preparing a FRET-based fluorescent probe according to claim 1 or 2, characterized in that: include: Quinolineboronic acid and a 1,2-dihydroxyanthraquinone derivative are mixed to obtain the FRET-based fluorescent probe.

4. The method for preparing a FRET-based fluorescent probe according to claim 3, wherein: The structure of the quinoline boronic acid is: The structure of the 1,2-dihydroxyanthraquinone derivative is: R is selected from one of H, sulfonic acid group and sulfonate.

5. The method for preparing a FRET-based fluorescent probe according to claim 3, wherein: The mixing is carried out in a solvent, and the solvent is selected from at least one of ethanol, methanol, tetrahydrofuran, acetone, water and acetonitrile.

6. A kit, characterized in that The invention comprises the FRET-based fluorescent probe according to claim 1 or the FRET-based fluorescent probe prepared by the preparation method according to claim 3.

7. The kit according to claim 6, characterized in that The kit further comprises a buffer solution and hexadecyltrimethylammonium bromide with a concentration of 2.0-15.0 mM, and the pH of the buffer solution is 4.0-6.

0.

8. Use of the FRET-based fluorescent probe according to claim 1, the FRET-based fluorescent probe prepared by the preparation method according to claim 3, or the kit according to claim 6 in detecting sialic acid, wherein the use does not involve the diagnosis and treatment of diseases.

9. A method for detecting sialic acid, characterized in that: The steps include: 1) The FRET-based fluorescent probe according to claim 1 or the FRET-based fluorescent probe prepared by the preparation method according to claim 3 is added to a buffer solution, and then a cetyltrimethylammonium bromide solution is added to the volume to obtain a detection system, and the fluorescence intensity at wavelengths of 400 nm and 580 nm is measured respectively, and the ratio of the fluorescence intensity at wavelengths of 400 nm to 580 nm is calculated. 400 / 580 ; 2) Add the test sample containing sialic acid to the detection system obtained in step 1), measure the fluorescence intensity at 400nm and 580nm wavelengths respectively, and calculate the ratio of the fluorescence intensity at 400nm to 580nm wavelengths F1 400 / 580 , through F1 400 / 580 / F0 400 / 580 The linear relationship with sialic acid concentration enables quantitative measurement of sialic acid test samples; The method does not involve the diagnosis and treatment of diseases.

10. The method for detecting sialic acid according to claim 9, wherein In the step 1), the volume ratio of the FRET-based fluorescent probe to the detection system is 15-25 μL:1 mL.

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