Rapid zinc content detection method based on fluorescence technology

Abstract

The invention discloses a fluorescence technology-based zinc content rapid detection method, which comprises: obtaining a to-be-detected zinc solution, a zinc standard solution and a cation-removed Zinpyr-1 test solution, adding the Zinpyr-1 test solution to the zinc standard solution, measuring the fluorescence intensity of the zinc standard solution, and calculating the fluorescence intensity of the zinc standard solution according to the fluorescence intensity of the Zinpyr-1 test solution. And drawing a relation curve of the corresponding zinc concentration and fluorescence intensity in the zinc standard solution, adding the Zinpyr-1 test solution into the to-be-detected zinc solution, detecting the fluorescence intensity of the to-be-detected zinc solution, and calculating the zinc concentration in the to-be-detected zinc solution according to the relation curve. The Zinpyr-1 fluorescent probe is used for detecting the content of zinc ions, high selectivity is achieved, interference of other metal ions is effectively avoided, and the detection sensitivity and resolution are improved.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, and in particular relates to a rapid method for detecting zinc content based on fluorescence technology. Background Technology

[0002] Commonly used quantitative detection methods for zinc include atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS).

[0003] Atomic absorption spectrometry (AAS) is a commonly used quantitative analysis method suitable for determining the zinc content in solutions. This method is based on the absorption phenomenon of a target element in a sample when it absorbs light of a specific wavelength. First, the sample is pretreated through steps such as gas combustion and high-temperature drying to convert it into a measurable form. Then, light corresponding to the wavelength of the target element is passed into the sample, and the intensity of the absorbed light is measured. This measurement is then compared with a zinc standard solution of known concentration to calculate the zinc content in the sample. Disadvantages: 1. Complex sample pretreatment and susceptibility to contamination (especially for trace analysis). 2. Susceptible to matrix interference, requiring careful optimization of conditions and background correction. 3. Low efficiency in single-element analysis. 4. Cannot provide speciation information (total zinc content in the measured sample).

[0004] Inductively coupled plasma mass spectrometry (ICP-MS) is a highly sensitive analytical technique suitable for determining the zinc content in liquid and solid samples. This method converts the sample into charged particles, which are then analyzed by mass spectrometry. ICP-MS offers a wide detection range and high resolution, allowing for the simultaneous determination of multiple elements. For zinc detection, ICP-MS provides a very low detection limit and high accuracy. Disadvantages include: 1. Low-mass zinc is susceptible to interference from overlapping peaks of other metal ions. 2. High-salt samples may suppress the signal, requiring dilution and purification. 3. High cost and complex daily maintenance.

[0005] Inductively coupled plasma optical emission spectrometry (ICP-OES) is a highly sensitive and selective analytical method widely used for multi-element analysis. It involves nebulizing the sample to form a plasma, and then measuring the specific wavelengths and intensities emitted by niobium in a spectrometer. Disadvantages include: 1. Spectral interference requires meticulous management; zinc's sensitive spectral lines are easily interfered with by overlapping spectral lines of elements such as Fe and Cr. 2. Strict sample pretreatment requirements: solid samples (soil, steel) must be digested into a solution, and the silicate structure must be completely destroyed; otherwise, the lattice-encapsulated zinc cannot be released. 3. Low salt requirements: salt concentrations >0.2% may clog the nebulizer or cone; high-salt samples require dilution or separation and purification. 4. High operating costs and complex maintenance. 5. Limited detection capability for light elements. Sensitivity is low for elements with a mass number <40 (such as As and Se), but zinc is not subject to this limitation. Note that co-occurring elements such as Cd and Hg may cause errors due to weak spectral lines.

[0006] Besides the methods mentioned above, other commonly used methods for zinc detection include electrochemical methods and spectrophotometry. Disadvantages: 1. Electrodes are easily contaminated; adsorption of organic matter or sulfides can lead to inaccurate signals. 2. Low sensitivity, unsuitable for trace analysis. 3. Severe interference. (Cd) 2+ Pb 2+ Cu 2+ It competes with dithizone for complexation (requiring precise control of masking agent dosage), and strong acids / bases can degrade the stability of the colorimetric reagent. 4. Reagent toxicity. Dithizone contains a cyano group, and carbon tetrachloride is carcinogenic; strict precautions are required. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to propose a method for detecting zinc content, addressing the shortcomings of existing zinc content detection methods mentioned in the background art.

[0008] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows: A rapid method for detecting zinc content based on fluorescence technology includes the following steps: Obtain the zinc solution to be tested, the zinc standard solution, and the Zinpyr-1 test solution after removing cations. Add the Zinpyr-1 test solution to the zinc standard solution, measure the fluorescence intensity of the zinc standard solution, and plot the relationship curve between the zinc concentration and the fluorescence intensity of the zinc standard solution. Add the Zinpyr-1 test solution to the zinc solution to be tested, measure the fluorescence intensity of the zinc solution to be tested, and calculate the zinc concentration in the zinc solution to be tested based on the relationship curve.

[0009] The design concept of the above technical solution is that Zinpyr-1, as a highly selective and sensitive fluorescent probe, is used for the detection and imaging of metal ions, especially zinc ions. The Zinpyr-1 molecule contains pyridine and other nitrogen-containing coordination sites, which can bind with Zn. 2+ It forms stable complexes and can preferentially bind Zn among numerous metal ions. 2+ This reduces interference signals. After the complex is formed, the electron transfer process within the molecule is regulated, leading to an enhancement or change in the fluorescence signal. This signal change can be detected by fluorescence spectroscopy or microscopic imaging equipment, enabling qualitative and quantitative analysis. Therefore, Zinpyr-1 can detect zinc ions down to nanomolar concentrations, making it suitable for trace element analysis in intracellular and biological samples. Zinpyr-1 and Zn 2+ The fluorescence intensity is significantly enhanced after binding, making it ideal for detecting extremely low concentrations of free zinc.

[0010] As a further preferred embodiment of the above technical solution, the zinc concentration in the zinc solution to be tested is x1, 0 < x1 < 0.023 μM, the volume of the zinc solution to be tested is L1, L1 ≤ 10 μL, and the concentration of the Zinpyr-1 test solution in the zinc solution to be tested is x2, and 0.5 μM < x2 < 5 μM.

[0011] As a further preferred embodiment of the above technical solution, the fluorescence intensity of the zinc solution to be tested is y1, and satisfies: y1 = -284315x1 2 +132314x1+32399.

[0012] As a further preferred embodiment of the above technical solution, the Zinpyr-1 test solution is a mixture of a cation-removing buffer solution, a balanced salt solution, and a Zinpyr-1 solution. The volume of the Zinpyr-1 test solution added to the zinc solution to be tested is L2, and L1+L2≤100μL.

[0013] As a further preferred embodiment of the above technical solution, the buffer solution removes cations by using an ion exchange resin and / or by adding sodium hydroxide solution, and the pH value of the buffer solution after cation removal is 7~7.5.

[0014] As a further preferred embodiment of the above technical solution, the concentration of the buffer solution is 50 mM; the buffer solution is an aqueous solution of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES).

[0015] As a further preferred embodiment of the above technical solution, the equilibrium salt solution is Hank's equilibrium salt solution (HBSS) and / or a 0.9% concentration saline solution; the Zinpyr-1 solution is prepared by dissolving Zinpyr-1 in 1% w / v dimethyl sulfoxide (DMSO), and the concentration of the Zinpyr-1 solution is 10 mM.

[0016] As a further preferred embodiment of the above technical solution, the Zinpyr-1 test solution is prepared by adding the balanced salt solution to the Zinpyr-1 solution to make the concentration of the Zinpyr-1 solution 10 μM, and then taking 10 μL of the Zinpyr-1 solution and adding it to 5 mL of the buffer solution to mix, thereby obtaining the Zinpyr-1 test solution.

[0017] As a further preferred embodiment of the above technical solution, the fluorescence intensity is obtained by measuring the fluorescence intensity using a fluorescence microplate reader at a temperature of 30℃~37℃ with an excitation wavelength of 495nm, an emission wavelength of 539nm, and a bandwidth of 5nm.

[0018] As a further preferred embodiment of the above technical solution, the rapid zinc content detection method based on fluorescence technology is used to detect the free zinc content in serum, cell / organoid culture medium or aqueous solution.

[0019] Compared with the prior art, the advantages of the present invention are as follows: This invention uses the Zinpyr-1 fluorescent probe to detect zinc ion content. The concentration of free zinc is quantified by measuring the change in fluorescence signal generated after zinc ions bind to the Zinpyr-1 fluorescent probe. This is because Zinpyr-1 and Zn... 2+ The fluorescence intensity is significantly enhanced after binding, making it ideal for detecting extremely low concentrations of free zinc. Zinpyr-1 also exhibits high selectivity for zinc ions, specifically binding to them and generating a fluorescence signal, effectively avoiding interference from other metal ions and ensuring accurate detection results. Furthermore, removing cations from the detection system avoids measurement deviations caused by changes in solution conductivity due to cations, and reduces background interference, improving detection sensitivity and resolution, making even weak signals easier to detect. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is the zinc ion concentration-fluorescence intensity relationship curve obtained in Example 1 of the present invention. Detailed Implementation

[0022] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0023] It should be noted that when a component is described as being "fixed to, attached to, connected to or connected to" another component, it can be directly fixed to, attached to, connected to or connected to the other component, or it can be indirectly fixed to, attached to, connected to or connected to the other component through other intermediate connectors.

[0024] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0025] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0026] Example 1: This embodiment describes a rapid zinc content detection method based on fluorescence technology, used to detect the free zinc content in a test sample (serum). The method includes the following steps: S1. Prepare Zinpyr-1 fluorescent probe working solution and zinc standard solution. Add Zinpyr-1 fluorescent probe working solution to zinc standard solution and test sample. Zinpyr-1 fluorescent probe working solution is prepared by the following method: first, dissolve Zinpyr-1 in DMSO (1% w / v) to prepare Zinpyr-1 stock solution, then dilute with Hankyr balanced salt solution to a Zinpyr-1 concentration of 10 μM. Then, take 10 μL of this mixed solution and add 5 mL of HEPES buffer and mix well to obtain Zinpyr-1 fluorescent probe working solution. HEPES buffer was prepared by the following method: The pH of a 50 mM aqueous solution of 4-hydroxyethylpiperazine ethanesulfonic acid was adjusted to 6 with sodium hydroxide solution to remove cations from the buffer, and then the pH was adjusted to 7 to obtain the HEPES buffer. The buffer was then subjected to adsorption treatment with Chelex-100 ion exchange resin to remove cations from the buffer. S2. Add Zinpyr-1 fluorescent probe working solution to 5 μL of zinc standard solutions of different concentrations to make the Zinpyr-1 fluorescent probe concentration in the detection system range from 0.5 μM to 5 μM. After mixing thoroughly, measure the fluorescence intensity to obtain a standard curve of zinc ion concentration versus fluorescence intensity. Figure 1 As shown; S3. Add Zinpyr-1 fluorescent probe working solution to 5 μL of the test sample to make the concentration of Zinpyr-1 fluorescent probe in the detection system 0.5 μM. After mixing evenly, measure the fluorescence intensity from 32642 to 46470. Calculate the zinc ion concentration in the test sample based on the standard curve of fluorescence intensity and zinc ion concentration-fluorescence intensity.

[0027] Zinpyr-1 is a highly selective and sensitive fluorescent probe used for the detection and imaging of metal ions, especially zinc ions. The Zinpyr-1 molecule contains pyridine and other nitrogen-containing coordination sites, which can bind to Zn. 2+ It forms stable complexes and can preferentially bind Zn among numerous metal ions. 2+This reduces interference signals. After the complex is formed, the electron transfer process within the molecule is regulated, leading to an enhancement or change in the fluorescence signal. This signal change can be detected by fluorescence spectroscopy or microscopic imaging equipment, enabling qualitative and quantitative analysis. Therefore, Zinpyr-1 can detect zinc ions down to nanomolar concentrations, making it suitable for trace element analysis in intracellular and biological samples. Zinpyr-1 and Zn 2+ The fluorescence intensity is significantly enhanced after binding, making it ideal for detecting extremely low concentrations of free zinc.

[0028] Furthermore, compared to large instruments such as ICP-MS, a fluorescence microplate reader is more economical and practical. Using the Zinpyr-1 fluorescent probe to detect zinc ion content, it has an extremely low detection limit, capable of detecting free zinc concentrations in the range of 0–0.023 μM. Simultaneously, it requires a small sample volume; only 6 μL of sample is needed for three repeated tests, representing a significant breakthrough compared to traditional ultrafiltration methods (mL-level). It also enables high-throughput detection; using a 96-well plate format, 20 samples can be analyzed simultaneously. With automated equipment, throughput can be further improved. In addition, the data obtained by this invention has excellent physiological relevance, detecting biologically active free zinc rather than total zinc, making it more directly related to various physiological and pathological processes.

[0029] The above are merely preferred embodiments of this application and are not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. For those skilled in the art, improvements and modifications obtained without departing from the technical concept of the present invention should also be considered within the protection scope of the present invention.

Claims

1. A rapid method for detecting zinc content based on fluorescence technology, characterized in that, Includes the following steps: Obtain the zinc solution to be tested, the zinc standard solution, and the Zinpyr-1 test solution after removing cations. Add the Zinpyr-1 test solution to the zinc standard solution, measure the fluorescence intensity of the zinc standard solution, and plot the relationship curve between the zinc concentration and the fluorescence intensity of the zinc standard solution. Add the Zinpyr-1 test solution to the zinc solution to be tested, measure the fluorescence intensity of the zinc solution to be tested, and calculate the zinc concentration in the zinc solution to be tested based on the relationship curve.

2. The rapid zinc content detection method based on fluorescence technology according to claim 1, characterized in that, The zinc concentration in the zinc solution to be tested is x1, where 0 < x1 < 0.023 μM, the volume of the zinc solution to be tested is L1, where L1 ≤ 10 μL, and the concentration of the Zinpyr-1 test solution in the zinc solution to be tested is x2, where 0.5 μM < x2 < 5 μM.

3. The method for detecting zinc content according to claim 2, characterized in that, The fluorescence intensity of the zinc solution to be tested is y1, and satisfies: y1 = -284315x1 2 +132314x1+32399.

4. The rapid zinc content detection method based on fluorescence technology according to claim 2, characterized in that, The Zinpyr-1 test solution is a mixture of a cation-removing buffer solution, a balanced salt solution, and a Zinpyr-1 solution. The volume of the Zinpyr-1 test solution added to the zinc solution to be tested is L2, and L1+L2≤100μL.

5. The rapid zinc content detection method based on fluorescence technology according to claim 4, characterized in that, The buffer solution removes cations by using an ion exchange resin and / or by adding sodium hydroxide solution, and the pH value of the buffer solution after cation removal is 7~7.

5.

6. The rapid zinc content detection method based on fluorescence technology according to claim 5, characterized in that, The concentration of the buffer solution is 50 mM; the buffer solution is an aqueous solution of 4-hydroxyethylpiperazine ethanesulfonic acid.

7. The rapid zinc content detection method based on fluorescence technology according to claim 4, characterized in that, The equilibrium salt solution is Hank's equilibrium salt solution and / or a 0.9% concentration of saline solution; the Zinpyr-1 solution is prepared by dissolving Zinpyr-1 in 1% w / v dimethyl sulfoxide, and the concentration of the Zinpyr-1 solution is 10 mM.

8. The rapid zinc content detection method based on fluorescence technology according to claim 7, characterized in that, The Zinpyr-1 test solution is prepared by adding the balanced salt solution to the Zinpyr-1 solution to make the concentration of the Zinpyr-1 solution 10 μM, and then taking 10 μL of the Zinpyr-1 solution and adding it to 5 mL of the buffer solution to obtain the Zinpyr-1 test solution.

9. The rapid zinc content detection method based on fluorescence technology according to any one of claims 1-8, characterized in that, Fluorescence intensity was measured using a fluorescence microplate reader at a temperature of 30℃~37℃ with an excitation wavelength of 495nm, an emission wavelength of 539nm, and a bandwidth of 5nm.

10. The rapid zinc content detection method based on fluorescence technology according to any one of claims 1-8, characterized in that, The rapid zinc content detection method based on fluorescence technology is used to detect the free zinc content in serum, cell / organoid culture medium or aqueous solution.

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