A nickel fluorescent nanocluster probe for biological detection and a preparation method and application thereof

Abstract

The application discloses a kind of histidine as preparation template with blue fluorescent emission ability nickel fluorescent nanoclusters His-NiNCs and its synthesis method and application, belong to chemical synthesis and biological analysis detection technical field.The nickel fluorescent nanoclusters has good water solubility and stability, with fluorescence emission ability, has wide application prospect in fluorescence sensing, biological imaging and other fields.The nickel fluorescent nanoclusters is prepared by simple aqueous chemical reduction synthesis method, and synthesis step is simple.Reaction time is short, and preparation raw material is cheap and easy to obtain.Based on the quenching effect of biological active substance horseradish peroxidase and hemoglobin on the fluorescence of the nickel fluorescent nanocluster probe, high-sensitivity analysis and detection of horseradish peroxidase and hemoglobin can be simultaneously realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of chemical synthesis and biological analysis detection, and relates to a preparation method of a nickel nanocluster probe for biological detection and application of the nickel nanocluster probe to detection of horseradish peroxidase and hemoglobin. BACKGROUND

[0002] Fluorescent metal nanoclusters (FMNCs) are small cluster-like nanomaterials formed by several to hundreds of 0-valence metal atoms stacked on an organic template molecule. Different from bulk metal materials, metal nanoparticles (MNPs), and metal cluster materials with a certain spatial skeleton structure, the particle size range of FMNCs is only about 1-5 nm. This ultra-small size is close to the Fermi wavelength of electrons, which can cause strong quantum confinement effect of free electrons in FMNCs, resulting in discrete energy level structure inside the FMNCs. The dispersed electron energy levels in FMNCs make them exhibit molecular-like electrical, optical and chemical properties, and excellent fluorescence emission performance is a major feature of FMNCs materials. In recent years, FMNCs materials have been widely used in fluorescence sensing, biological analysis, environmental analysis, biological imaging, and virus and nucleic acid detection due to their high luminescence intensity, good luminescence stability, large Stokes shift, strong anti-interference ability, low biological toxicity, small size, and easy to penetrate cells. Multifunctional and multi-modal sensing and detection probes based on FMNCs materials have broad application prospects and market space.

[0003] Currently, the preparation methods of FMNCs mainly include induced etching method ("top-down" method), aqueous chemical reduction method, hydrothermal / solvothermal method, and electrochemical reduction method (collectively referred to as "bottom-up" method). Compared with other preparation methods, the aqueous chemical reduction method using organic ligands as a preparation template has the advantages of simplicity, low energy consumption, no need for corrosive reagents, and environmental friendliness, and is most widely used in the preparation of FMNCs materials. However, according to the redox characteristics of metals, it is difficult to directly reduce the cations of metal elements after hydrogen to 0-valence metal atoms in aqueous solution at normal temperature and pressure. Therefore, under the current technical conditions, FMNCs prepared by the aqueous chemical reduction method are mostly made of metals such as gold, silver, copper, and platinum after hydrogen, and it is more difficult to prepare FMNCs from metals such as iron, manganese, and nickel before hydrogen, which leads to a small number of types of existing FMNCs materials and single function, and the existing FMNCs materials cannot meet the application needs of society and market for high-performance and multifunctional FMNCs materials. It is urgent to expand the types and functions of FMNCs materials and develop new transition metal fluorescent nanocluster materials.

[0004] In view of the deficiencies of the prior art, the application discloses a nickel fluorescent nanocluster (His-Ni NCs) probe with blue fluorescence emission capacity and taking histidine as a template, and provides a rapid, efficient, green and low-cost aqueous-phase reduction preparation method for preparing the His-Ni NCs probe. Compared with noble metal elements such as gold and silver, nickel element is widely available, cheap and easy to obtain, and has magnetism, which is an ideal choice for developing multifunctional FMNCs materials. The His-Ni NCs probe disclosed by the application belongs to hydrogen pre-metal fluorescent nanoclusters, has good water solubility and stability, and the fluorescence emitted thereby can be quenched by horseradish peroxidase and hemoglobin. Accordingly, the His-Ni NCs probe is developed into a fluorescence probe for detecting horseradish peroxidase and hemoglobin and applied to the field of life analysis, and has obvious technical progress and wide application prospect. SUMMARY

[0005] The application aims to overcome the deficiencies of the prior art, and provides a water-soluble blue light emitting nickel fluorescent nanocluster (His-Ni NCs) probe prepared from hydrogen pre-metal Ni and a simple aqueous-phase chemical reduction synthesis method thereof. The His-Ni NCs probe has excellent blue fluorescence emission capacity and fluorescence sensing response capacity to horseradish peroxidase and hemoglobin, and has good application prospect in protein detection.

[0006] To achieve the above object, the application discloses a blue light emitting nickel fluorescent nanocluster probe His-Ni NCs, which is characterized by:

[0007] (1) is prepared as follows:

[0008] 1) histidine is selected as a preparation template.

[0009] 2) a certain amount of histidine solid is dissolved in high-purity water to prepare a histidine solution, and a certain amount of Ni 2+ salt solution is added dropwise to the histidine solution, and the mixture is stirred uniformly at room temperature to form a reaction precursor solution.

[0010] 3) a certain amount of ascorbic acid solid is added to the reaction precursor solution, and the ascorbic acid is dissolved by fully stirring, and then a certain amount of NaOH solution is added dropwise to adjust the pH of the reaction system.

[0011] 4) the reaction mixture is stirred at room temperature for a certain period of time until the solution becomes light pink, and then the nickel fluorescent nanocluster His-Ni NCs is obtained.

[0012] (2) the nickel fluorescent nanocluster probe has a near-spherical shape, a particle size range of 1.2-3.2 nm, and an average particle size of 2.0 nm.

[0013] (3) The nickel fluorescent nanocluster probe has the ability to emit blue fluorescence, and the maximum excitation wavelength is located at 352 nm, and the maximum emission wavelength is located at 420 nm.

[0014] (4) The nickel nanocluster probe has the ability of fluorescent sensing response to horseradish peroxidase and hemoglobin at the same time, and the fluorescence can be quenched by the above two target objects.

[0015] The application further discloses a synthesis method of the nickel fluorescent nanocluster probe His-Ni NCs, and the synthesis method is performed according to the following steps:

[0016] (1) A certain amount of histidine solid is dissolved in high-purity water to prepare a histidine solution with a concentration ranging from 0.015 to 0.020 M, and a certain amount of 0.1 M Ni 2+ salt solution is added dropwise into the histidine solution, the molar ratio of Ni 2+ and histidine is 1:9.5, and the reaction precursor solution is uniformly stirred at room temperature.

[0017] (2) A certain amount of ascorbic acid solid is added into the reaction precursor solution, the molar ratio of the added ascorbic acid and Ni 2+ in the reaction precursor solution is 19:1, the ascorbic acid is fully stirred and dissolved, and a certain amount of NaOH solution is added to adjust the pH value of the reaction system, and the molar ratio of the added NaOH and Ni 2+ in the reaction precursor solution is 4:3.

[0018] (3) The reaction mixture is stirred at room temperature for 3-5 h until the solution becomes light pink, and the nickel fluorescent nanocluster His-Ni NCs is obtained.

[0019] The Ni 2+ salt solution selected in the step (1) can be any aqueous solution of inorganic salt formed by matching anions with Ni 2+ .

[0020] The application further discloses application of the His-Ni NCs probe prepared by the method in detection of horseradish peroxidase in a solution. Specifically, a certain volume of His-Ni NCs probe solution, peroxidase standard solution and high-purity water are taken to prepare a detection system, the quenching amount (ΔF) of the fluorescent signal of the Nis-Ni NCs probe to the target horseradish peroxidase is determined by using the detection system containing different concentrations of horseradish peroxidase standard solution, and the concentration of the horseradish peroxidase in a sample is determined according to the standard curve with the quenching amount (ΔF) as the vertical coordinate and the concentration of the horseradish peroxidase standard solution as the horizontal coordinate.

[0021] In the detection method, the linear equation is ΔF=26.40x+53.49, ΔF=F0-F, F0 is the fluorescence intensity of the blank probe solution without the addition of horseradish peroxidase, F is the fluorescence intensity of the probe test solution after the addition of horseradish peroxidase, x is the concentration of horseradish peroxidase, and the linear correlation coefficient R 2 =0.9958.

[0022] In the detection method, the linear range of the His-Ni NCs probe for the detection of horseradish peroxidase is 0.65 μM-9 μM, and the detection limit is 38 nM.

[0023] In the detection method, the test solution contains 3.80 mL of the His-Ni NCs probe solution diluted by 10 times and 0.20 mL of the reduced glutathione standard solution; and the blank solution contains the same volume of high-purity water instead of the horseradish peroxidase standard solution.

[0024] In the detection method, the fluorescence intensity of the test solution is detected after the addition of the horseradish peroxidase standard solution into the His-Ni NCs probe solution and the sufficient reaction for 20 min.

[0025] The application further discloses application of the His-Ni NCs probe prepared by the method in detection of hemoglobin in a solution.

[0026] In the detection method, the linear equation is ΔF=7.67x+58.38, ΔF=F0-F, F0 is the fluorescence intensity of the blank probe solution without the addition of hemoglobin, F is the fluorescence intensity of the probe test solution after the addition of hemoglobin, x is the concentration of hemoglobin, and the linear correlation coefficient R 2 =0.9949.

[0027] In the detection method, the linear range of the His-Ni NCs probe for the detection of hemoglobin is 3-25 μM, and the detection limit is 0.206 μM.

[0028] In the detection method, the test solution contains 3.80 mL of the His-Ni NCs probe solution diluted by 12 times and 0.20 mL of the hemoglobin solution; and the blank solution contains the same volume of high-purity water instead of the hemoglobin standard solution.

[0029] In the above detection method, the fluorescence intensity of the test solution is detected after the hemoglobin standard solution is added to the His-Ni NCs probe solution and fully reacted for 15 min.

[0030] The blue light-emitting fluorescent His-Ni NCs probe and the synthesis method thereof disclosed by the application have the following positive effects compared with the prior art:

[0031] (1) The nickel fluorescent nanoclusters synthesized by the application have stable fluorescence properties, and the obtained product has small particle size and good biocompatibility; the synthesis method of the probe is simple and fast, and does not need additional steps such as functional modification.

[0032] (2) The application can be used for simple, fast, economical, sensitive and highly selective detection of horseradish peroxidase in a solution at room temperature, and the linear range for detection of horseradish peroxidase is 0.65-9 μM, and the detection limit is 38 nM.

[0033] (3) The application can be used for simple, fast, economical, sensitive and highly selective detection of hemoglobin in a solution at room temperature, and the linear range for detection of hemoglobin is 3-25 μM, and the detection limit is 0.206 μM. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The TEM (transmission electron microscope) image of His-Ni NCs shows that the particle size of the synthesized His-Ni NCs is uniform and small;

[0035] Figure 2 The particle size distribution diagram of His-Ni NCs shows the particle size range and average particle size thereof.

[0036] Figure 3 The XPS (X-ray photoelectron spectroscopy) wide spectrum diagram of His-Ni NCs shows the main element composition thereof;

[0037] Figure 4 The fluorescence excitation spectrum and emission spectrum diagram of His-Ni NCs shows that the maximum excitation wavelength is 352 nm and the maximum emission wavelength is 420 nm;

[0038] Figure 5 The standard curve diagram of His-Ni NCs for detection of horseradish peroxidase shows that the His-Ni NCs probe can be successfully applied to high-sensitivity fluorescence detection of horseradish peroxidase, and has good application prospect in biological analysis.

[0039] Figure 6The standard curve of His-Ni NCs for detecting hemoglobin shows that the His-Ni NCs probe can be successfully applied to high-sensitivity fluorescence detection of the target hemoglobin, and has good application prospects in biological analysis. DETAILED DESCRIPTION

[0040] The present application is described below through specific embodiments. Unless specifically stated, the technical means used in the present application are methods known to those skilled in the art. In addition, the embodiments are understood to be illustrative, rather than limiting the scope of the present application, and the essence and scope of the present application are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and amounts in these embodiments without departing from the essence and scope of the present application also fall within the protection scope of the present application.

[0041] The reagents used are all analytical pure, and the reagents used and the manufacturers are as follows: high-purity water, Hangzhou Wahaha Group Co., Ltd.; histidine, Shenguo Biological (Shanghai) Co., Ltd.; nickel chloride, Shanghai Titan Technology Co., Ltd.; ascorbic acid, Tianjin Kemio Reagent Co., Ltd.; sodium hydroxide, Tianjin Fuchan Chemical Reagent Co., Ltd.; horseradish peroxidase, Nanjing Jiancheng Biological Technology Co., Ltd.; hemoglobin, Shenguo Biological (Shanghai) Co., Ltd.

[0042] Example 1

[0043] The aqueous-phase chemical reduction preparation of nickel fluorescent nanoclusters (His-Ni NCs) with histidine (His) as a preparation template is carried out according to the following steps:

[0044] (1) 0.0441 g of histidine was weighed and dissolved in 15 mL of high-purity water to prepare a histidine solution with a concentration of 0.019 M, and 300 μL of a 0.1 M NiCl2 solution was added dropwise to the solution. At this time, the molar ratio of Ni 2+ to histidine in the reaction system was 1:9.5, and the reaction mixture solution was stirred uniformly at room temperature.

[0045] (2) 0.1 g of ascorbic acid was weighed and added to the above reaction mixture solution and stirred thoroughly. At this time, the molar ratio of ascorbic acid to Ni 2+ was 19:1.

[0046] (3) 400 μL of a 1 M NaOH solution was immediately added dropwise to the above reaction mixture solution. At this time, the molar ratio of NaOH to Ni 2+ was 4:3, and the above reaction mixture solution was stirred and mixed at room temperature for 4.5 h until the solution turned light pink, and His-Ni NCs were obtained.

[0047] Example 2

[0048] (1) The preparation method of His-Ni NCs refers to Example 1;

[0049] (2) Transmission electron microscopy (TEM) characterization of His-Ni NCs:

[0050] The prepared His-Ni NCs were dispersed in high-purity water, uniformly dropped on a copper mesh, dried, and the observation sample was prepared. The morphology of His-Ni NCs was observed by field emission transmission electron microscopy. As shown in Figure 1 , the morphology of His-Ni NCs is nearly spherical and uniformly dispersed; the particle size of His-Ni NCs in the TEM image was counted, Figure 2 showing that the particle size ranges from 1.2 to 3.2 nm, the particle size distribution is relatively uniform, and the average particle size of His-Ni NCs is 2.0 nm.

[0051] Example 3

[0052] (1) The preparation method of His-Ni NCs refers to Example 1;

[0053] (2) Elemental composition characterization of His-Ni NCs:

[0054] After the prepared His-Ni NCs were purified and dried, X-ray photoelectron spectroscopy (XPS) characterization was performed, and the XPS wide spectrum is shown in Figure 3 . The experimental results show that the His-Ni NCs are composed of elements such as C, O, and Ni, and the O and C elements come from the template histidine.

[0055] Example 4

[0056] (1) The preparation method of His-Ni NCs refers to Example 1;

[0057] (2) Determination of fluorescence excitation spectrum and emission spectrum of His-Ni NCs:

[0058] The prepared His-Ni NCs were dispersed in high-purity water, and the fluorescence excitation spectrum and emission spectrum of the His-Ni NCs sample were determined by fluorescence spectrophotometry, as shown in Figure 4 , the maximum excitation wavelength of the His-Ni NCs is 352 nm, and under the excitation of this maximum excitation wavelength, the fluorescence emission wavelength is 420 nm.

[0059] Example 5

[0060] The application of His-Ni NCs probe in horseradish peroxidase (HRP) detection was carried out according to the following steps:

[0061] (1) The preparation method of His-Ni NCs refers to Example 1;

[0062] (2) The prepared His-Ni NCs probe stock solution is diluted 10 times;

[0063] (3) A series of horseradish peroxidase standard solutions are respectively prepared;

[0064] (4) 3800 μL of the diluted His-Ni NCs probe solution and 200 μL of high-purity water are added to a 5 mL centrifuge tube, shaken well, and after 20 min, a blank detection solution is prepared;

[0065] (5) Another 5 mL centrifuge tube is taken, 200 μL of the reduced horseradish peroxidase solution prepared in step (3) and 3800 μL of the diluted His-Ni NCs probe solution in step (2) are added respectively, shaken well and reacted for 20 min, then a sample detection solution is prepared;

[0066] (6) The fluorescence intensity F0 of the above blank detection solution and the fluorescence intensity F of the sample detection solution are detected by a fluorescence spectrophotometer respectively, the difference between the two, i.e. the quenching amount of horseradish peroxidase on the fluorescence signal of His-Ni NCs probe (ΔF=F0-F), is taken as the vertical coordinate, and the concentration of horseradish peroxidase standard solution is taken as the horizontal coordinate to draw a standard curve, and the concentration of horseradish peroxidase in the sample is determined according to the standard curve.

[0067] Figure 5 The experimental results shown in the table show that when the concentration of horseradish peroxidase is in the range of 0.65-9 μM, the fluorescence intensity quenching value of His-Ni NCs probe presents a linear relationship with the concentration of horseradish peroxidase, the linear equation is F0-F=26.40x+53.49, the linear correlation coefficient is 0.9958, and the detection limit is 38 nM.

[0068] Example 6

[0069] The application of His-Ni NCs probe in hemoglobin (Hb) detection is carried out according to the following steps:

[0070] (1) The preparation method of His-Ni NCs refers to Example 1;

[0071] (2) The prepared His-Ni NCs probe stock solution is diluted 12 times;

[0072] (3) A series of hemoglobin standard solutions are respectively prepared;

[0073] (4) 3800 μL of the diluted His-Ni NCs probe solution and 200 μL of high-purity water are added to a 5 mL centrifuge tube, shaken well, and after 20 min, a blank detection solution is prepared;

[0074] (5) Another 5 mL centrifuge tube, respectively, to which 200 μL prepared in step (3) of the hemoglobin standard solution, and 3800 μL diluted in step (2) His-Ni NCs probe solution, after 15 min, prepared sample detection solution;

[0075] (6) The fluorescence spectrophotometer was used to detect the fluorescence intensity F0 and the fluorescence intensity F of the sample detection solution, respectively. The difference between the two, that is, the quenching amount of hemoglobin on the His-Ni NCs probe fluorescence signal (ΔF = F0-F) was taken as the vertical coordinate, and the concentration of hemoglobin standard solution was taken as the horizontal coordinate to draw the standard curve. The concentration of hemoglobin in the sample was determined according to the standard curve.

[0076] Figure 6 The experimental results show that the fluorescence intensity quenching amount of His-Ni NCs probe and the concentration of hemoglobin present a linear relationship in the range of 3-25 μM of hemoglobin concentration. The linear equation is F0-F = 7.67x+58.38, the linear correlation coefficient is 0.9949, and the detection limit is 0.206 μM.

Claims

1. Application of blue light-emitting nickel fluorescent nanocluster probe His-Ni NCs in high-sensitivity detection of horseradish peroxidase, characterized in that: The blue light-emitting nickel fluorescent nanocluster probe His-Ni NCs used are prepared according to the following steps: (1) selecting histidine as a preparation template; (2) A certain amount of histidine solid is dissolved in high-purity water to obtain a histidine solution, and a certain amount of Ni 2+ salt solution is added dropwise to the histidine solution, and the mixture is stirred uniformly at room temperature to form a reaction precursor solution; (3) adding a certain amount of solid ascorbic acid into the above reaction precursor solution, fully stirring to dissolve the ascorbic acid, and then adding a certain amount of NaOH solution to adjust the pH of the reaction system; (4) stirring the above reaction mixture at room temperature for a certain period of time until the solution turns light pink, and nickel fluorescent nanoclusters His-Ni NCs are obtained; The nickel fluorescent nanocluster probe has the ability to emit blue fluorescence, with a maximum excitation wavelength of 352 nm and a maximum emission wavelength of 420 nm.

2. Application of blue light-emitting nickel fluorescent nanocluster probe His-Ni NCs in high-sensitivity detection of hemoglobin, characterized in that: The blue light-emitting nickel fluorescent nanocluster probe His-Ni NCs used are prepared according to the following steps: (1) selecting histidine as a preparation template; (2) A certain amount of histidine solid is dissolved in high-purity water to prepare a histidine solution, and a certain amount of Ni 2+ salt solution is added dropwise to the histidine solution, and the mixture is stirred uniformly at room temperature to form a reaction precursor solution; (3) adding a certain amount of solid ascorbic acid into the above reaction precursor solution, fully stirring to dissolve the ascorbic acid, and then adding a certain amount of NaOH solution to adjust the pH of the reaction system; (4) stirring the above reaction mixture at room temperature for a certain period of time until the solution turns light pink, and nickel fluorescent nanoclusters His-Ni NCs are obtained; The nickel fluorescent nanocluster probe has the ability to emit blue fluorescence, with a maximum excitation wavelength of 352 nm and a maximum emission wavelength of 420 nm.

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