阅读说明：本技术 一种石蒜碱作为检测羟自由基荧光探针的测定方法 (Determination method of lycorine as fluorescent probe for detecting hydroxyl radicals ) 是由 朱彧 张斌 王远宏 徐立婧 张丽霞 于 2019-08-16 设计创作，主要内容包括：本发明公开了一种石蒜碱作为检测羟自由基荧光探针的测定方法,通过石蒜碱对羟自由基的荧光响应,以及石蒜碱的荧光强度与加入的羟自由基量的关系,能够对微摩尔级的羟自由基进行精确定量。具体包括以下步骤：首先确定石蒜碱的激发与发射波长,然后对石蒜碱进行不同离子,不同浓度过氧化氢和不同浓度羟自由基的荧光响应检测,得出1.176μM的石蒜碱对过氧化氢与硫酸亚铁产生的羟自由基有响应,可以作为羟自由基的荧光探针,其对过氧化氢没有荧光响应,不能够作为过氧化氢的荧光探针及石蒜碱的荧光强度与所加的羟自由基浓度存在着二次函数关系,说明石蒜碱可以作为羟自由基的荧光探针,检测很灵敏,可以对微摩尔级的羟自由基进行精确地定量。(The invention discloses a method for measuring lycorine as a fluorescent probe for detecting hydroxyl radicals, which can accurately quantify the hydroxyl radicals in micromolar level through the fluorescent response of lycorine to the hydroxyl radicals and the relation between the fluorescent intensity of lycorine and the amount of the added hydroxyl radicals. The method specifically comprises the following steps: firstly, determining the excitation and emission wavelengths of lycorine, then carrying out fluorescence response detection on lycorine with different ions, hydrogen peroxide with different concentrations and hydroxyl radicals with different concentrations to obtain that 1.176 mu M of lycorine has response to the hydroxyl radicals generated by the hydrogen peroxide and ferrous sulfate, can be used as a fluorescent probe of the hydroxyl radicals, has no fluorescence response to the hydrogen peroxide, cannot be used as the fluorescent probe of the hydrogen peroxide, and has a quadratic function relationship between the fluorescence intensity of the lycorine and the concentration of the added hydroxyl radicals, which indicates that the lycorine can be used as the fluorescent probe of the hydroxyl radicals, has sensitive detection and can accurately quantify the hydroxyl radicals at micromolar level.)

1. A method for measuring lycorine as a fluorescent probe for detecting hydroxyl radicals is characterized by comprising the following steps: the method comprises the following steps:

(1) determining the excitation and emission wavelengths of lycorine: detecting with a fluorescence spectrophotometer, adding 3200 μ L of lycoris alkali solution with concentration of 0.6623 μ M into a four-side light colorimetric dish, setting parameters, selecting an excitation wavelength of 360nm, scanning wavelength range of 380-640nm, finding out that the maximum excitation wavelength of lycorine is 248nm and the maximum emission wavelength is 410nm,

(2) the detection of different ion fluorescence responses to lycorine: setting the determined excitation wavelength as a fixed value, starting scanning within a scanning range of 258-478 nm, wherein the obtained curve is the fluorescence curve of the original lycorine solution, sequentially adding 20 μ L of 10mM calcium chloride, potassium chloride, magnesium sulfate, sodium hypochlorite, hydrogen peroxide and ferrous sulfate solution into 1.176 μ M lycorine solution, scanning to obtain fluorescence response diagrams of different ions to the lycorine solution, storing data,

(3) and (3) detecting the fluorescence response of hydrogen peroxide with different concentrations to lycorine: adding 3400 μ L of lycorine solution with concentration of 1.176 μ M into a four-side light colorimetric cuvette, setting excitation wavelength at 248nm, scanning within 258-478 nm, scanning to obtain lycorine original solution curve, sequentially adding 20 μ L of 10 μ M, 200 μ M, 500 μ M, 50mM, 80mM, 100mM, and 500mM hydrogen peroxide solution into the colorimetric cuvette, scanning to obtain fluorescence response graphs of different concentrations to lycorine solution, storing the data obtained by scanning,

(4) and (3) detecting the fluorescence response of hydroxyl radicals with different concentrations to lycorine: hydroxyl radical is generated by the reaction of 3 percent hydrogen peroxide and ferrous sulfate, the lycorine raw solution is firstly scanned, 3400 mu L of 1.176 mu M lycorine solution with the concentration is added into a four-side light colorimetric dish, the excitation wavelength is set to be 248nm, the scanning range is set to be 258nm-478nm, the fluorescence curve of the lycorine raw solution is obtained, 20 mu L of 10mM hydrogen peroxide is firstly added, the scanning is started, the scanning is waited for 2-5 minutes after the scanning is finished, the scanning is carried out again until the fluorescence curve is stable and does not change any more, and the added hydrogen peroxide curve is deleted; then adding 20 mu L2.5mM ferrous sulfate solution, starting scanning, wherein the obtained curve is a fluorescence response graph of 0.1162mM hydroxyl free radicals to lycoris radiata alkali solution after 5s, scanning again after waiting for 5 minutes, and recording and storing the curve; continuously adding 20 mu L of 10mM hydrogen peroxide for scanning, observing the curve until the curve is not changed any more and then deleting the curve; and adding 20 mu L of 10mM ferrous sulfate solution, scanning to obtain a fluorescence response graph of 0.1449mM hydroxyl free radical to lycoris radiata alkali solution for 5s and 5min, not adding any more until the concentration of the added hydroxyl free radical is 0.3954mM, and recording and storing the obtained graphs and data of 5s and 5min for later data analysis.

2. The method for detecting lycorine as a fluorescent probe for detecting hydroxyl radicals according to claim 1, which is characterized in that: in step (2), to verify the fluorescent response of different ions to lycorine, 20. mu.L of KCl, MgSO 5. mu.M was added at a concentration of 5.8. mu.L₄5.7 μ M CaCl₂、NaClO、H₂O₂Adding into lycorine containing 1.176 μ M, and using 248nm as EX and EM as 414nm, respectively, obtaining no change in fluorescence intensity at maximum emission wavelength of 414nm from fluorescence spectrum change chart, and adding FeSO with concentration of 5.6 μ M of 20 μ M₄The fluorescence intensity decreased, and the fluorescence intensity did not change when 20. mu.L of 5.7. mu.M hydrogen peroxide was added, and began to decrease again when 20. mu.L of 5.6. mu.M FeSO4 was added, because lycorine fluoresced in response to hydroxyl radicals generated from hydrogen peroxide and ferrous sulfate.

3. The method for detecting lycorine as a fluorescent probe for detecting hydroxyl radicals according to claim 1, which is characterized in that: in step (3), in order to verify the fluorescent response of lycorine to hydrogen peroxide, 20 μ L of hydrogen peroxide at different concentrations was sequentially added to a solution containing 1.176 μ M of lycorine, with 248nm as EX and 414nm as EM, and it was observed that the fluorescence intensity of lycorine decreased slightly but changed slightly from that without hydrogen peroxide as the concentration of hydrogen peroxide was added.

4. The method for detecting lycorine as a fluorescent probe for detecting hydroxyl radicals according to claim 1, which is characterized in that: in step (4), as shown by the graph of the change of the fluorescence spectrum after 5 minutes, in lycorine having a concentration of 1.176. mu.M, with EX at 248nm and 411nm as EM, the maximum emission peak appears at 414nm before hydroxyl radical is not added, and when the concentration of added hydroxyl radical is 0.1162mM, a new peak appears at the beginning of EM at 364nm, the fluorescence intensity of the new emission peak is 100nm, the fluorescence intensity of lycorine is continuously increased with the continuous increase of the concentration of added hydroxyl radical, and when the amount of added hydroxyl radical reaches 0.3954mM, the fluorescence intensity of the maximum emission peak at 364nm is increased by 8 times, the increase of the fluorescence intensity of lycorine is closely related to the concentration of added hydroxyl radical, and there is a quadratic function relationship between the concentration of 0.1449mM and 0.3683 mM.

Technical Field

The invention belongs to the technical field of fluorescent probes, and particularly relates to a determination method of lycorine as a fluorescent probe for detecting hydroxyl radicals.

Background

At present, methods for detecting cellular active oxygen mainly include an electron spin resonance method, a high performance liquid chromatography, an ultraviolet spectrophotometer method and a fluorescence method. The fluorescent probe detection method is one of fluorescent methods, and is mainly characterized in that a probe with specific receptor binding is used for detection, the probe with specific receptor binding is identified with a target substance, so that the molecular structure of the probe is changed, the fluorescent property of the probe is further changed, information of the target molecule is obtained, and the target substance molecule becomes a detectable fluorescent signal by means of a fluorescent imaging technology, so that visual detection in a living body or in an environment is achieved. The fluorescent probe detection technology has high sensitivity, high activity and convenient application, and the fluorescent probe can enter cells to be combined with active oxygen in the cells, so the fluorescent probe is mainly applied to the aspects of medicine and the like.

Disclosure of Invention

The invention aims to solve the problem of providing a method for measuring lycorine as a fluorescent probe for detecting hydroxyl radicals, which can accurately quantify the hydroxyl radicals in micromolar level through the fluorescent response of lycorine to the hydroxyl radicals and the relation between the fluorescent intensity of lycorine and the amount of the added hydroxyl radicals.

In order to solve the technical problems, the invention adopts the technical scheme that: a method for measuring lycorine as a fluorescent probe for detecting hydroxyl radicals is characterized by comprising the following steps: the method comprises the following steps:

(1) determining the excitation and emission wavelengths of lycorine: detecting with a fluorescence spectrophotometer, adding 3200 μ L of lycorine solution with concentration of 0.6623 μ M into a four-side light colorimetric dish, initially setting parameters, selecting an excitation wavelength of 360nm (initial setting, and then precisely selecting the excitation wavelength), setting the scanning wavelength range to be 380-640nm (the scanning initial wavelength should be 10-20nm longer than the excitation wavelength, and the scanning end wavelength should be 2 times shorter than the excitation wavelength to avoid the frequency doubling peak of the instrument), finding out that the maximum excitation wavelength of lycorine is 248nm and the maximum emission wavelength is 410nm,

(2) the detection of different ion fluorescence responses to lycorine: setting the determined excitation wavelength as a fixed value, starting scanning within a scanning range of 258-478 nm, wherein the obtained curve is the fluorescence curve of the original lycorine solution, sequentially adding 20 μ L of 10mM calcium chloride, potassium chloride, magnesium sulfate, sodium hypochlorite, hydrogen peroxide and ferrous sulfate solution into 1.176 μ M lycorine solution, scanning to obtain fluorescence response diagrams of different ions to the lycorine solution, storing data,

(3) and (3) detecting the fluorescence response of hydrogen peroxide with different concentrations to lycorine: adding 3400 μ L of lycorine solution with concentration of 1.176 μ M into a four-side light colorimetric cuvette, setting excitation wavelength at 248nm, scanning within 258-478 nm, scanning to obtain lycorine original solution curve, sequentially adding 20 μ L of 10 μ M, 200 μ M, 500 μ M, 50mM, 80mM, 100mM, and 500mM hydrogen peroxide solution into the colorimetric cuvette, scanning to obtain fluorescence response graphs of different concentrations to lycorine solution, storing the data obtained by scanning,

(4) and (3) detecting the fluorescence response of hydroxyl radicals with different concentrations to lycorine: hydroxyl radical is generated by the reaction of 3% hydrogen peroxide and ferrous sulfate, the lycorine raw solution is firstly scanned, 3400 mu L of 1.176 mu M lycorine solution with the concentration is added into a four-side light colorimetric dish, the excitation wavelength is set to be 248nm, the scanning range is set to be 258nm-478nm, the fluorescence curve of the lycorine raw solution is obtained, 20 mu L of 10mM hydrogen peroxide is firstly added, scanning is started, scanning is carried out again after 2-5 minutes after the scanning is finished until the fluorescence curve is stable and does not change any more (the error caused by the hydrogen peroxide is reduced), and the added hydrogen peroxide curve is deleted; then adding 20 mu L2.5mM ferrous sulfate solution, starting scanning, wherein the obtained curve is a fluorescence response graph of 0.1162mM hydroxyl free radicals to lycoris radiata alkali solution after 5s, scanning again after waiting for 5 minutes, and recording and storing the curve; continuously adding 20 mu L of 10mM hydrogen peroxide for scanning, observing the curve until the curve is not changed any more and then deleting the curve; and adding 20 mu L of 10mM ferrous sulfate solution, scanning to obtain a fluorescence response graph of 0.1449mM hydroxyl free radical to lycoris radiata alkali solution for 5s and 5min, not adding any more until the concentration of the added hydroxyl free radical is 0.3954mM, and recording and storing the obtained graphs and data of 5s and 5min for later data analysis.

In step (2), to verify the fluorescent response of different ions to lycorine, 20. mu.L of KCl, MgSO 5. mu.M was added at a concentration of 5.8. mu.L₄5.7 μ M CaCl₂、NaClO、H₂O₂Adding into lycorine containing 1.176 μ M, and using 248nm as EX (excitation wavelength) and EM (emission wavelength) as 414nm, wherein the fluorescence intensity at maximum emission wavelength of 414nm is unchanged from fluorescence spectrum change chart, and when adding FeSO with 20 μ M concentration of 5.6 μ M₄The fluorescence intensity was reduced because hydroxyl radicals were generated by the reaction of added hydrogen peroxide and ferrous sulfate, the fluorescence intensity was unchanged when 20. mu.L of hydrogen peroxide was added at a concentration of 5.7. mu.M, and the fluorescence intensity began to decrease when 20. mu.L of FeSO4 was added at a concentration of 5.6. mu.M, because lycorine fluoresced in response to hydroxyl radicals generated by hydrogen peroxide and ferrous sulfate.

In step (3), in order to verify the fluorescent response of lycorine to hydrogen peroxide, 20 μ L of hydrogen peroxide at different concentrations was sequentially added to a solution containing 1.176 μ M of lycorine, with 248nm as EX (excitation wavelength) and EM (emission wavelength) as 414nm, and it was observed that the intensity of fluorescence of lycorine decreased slightly, but slightly, from that of fluorescence without addition of hydrogen peroxide, depending on the concentration of hydrogen peroxide.

In step (4), as a result of a change in fluorescence spectrum after 5 minutes, in lycorine having a concentration of 1.176. mu.M, 248nm is EX (excitation wavelength), EM (emission wavelength) is 411nm, the maximum emission peak appears at 414nm before hydroxyl radical is not added, and when the concentration of added hydroxyl radical is 0.1162mM, a new peak begins to appear at 364nm with EM, the fluorescence intensity of the new emission peak is 100nm, the fluorescence intensity of lycorine is continuously increased as the concentration of added hydroxyl radical is continuously increased, and when the amount of added hydroxyl radical reaches 0.3954mM, the fluorescence intensity of the maximum emission peak at 364nm is increased by 8 times, the increase in fluorescence intensity of lycorine is closely related to the concentration of added hydroxyl radical, and there is a quadratic function relationship between the concentration of 0.1449mM and 0.3683 mM.

Due to the adoption of the technical scheme, (1) the invention contains 1.176 mu MAdding 20 μ L of KCl and MgSO 5.8 μ M₄5.7 μ M CaCl₂、NaClO、H₂O₂There was no change in fluorescence intensity when 20. mu.L of 5.6. mu.M FeSO was added₄While the fluorescence intensity at 414nm of the maximum emission peak was reduced, 20. mu.L of 5.7. mu.MH was added₂O₂The fluorescence intensity did not change until 20. mu.L of 5.6. mu.M FeSO was added₄The fluorescence intensity begins to decrease again, so that the 1.176 mu M lycorine can be obtained to respond to the hydroxyl radical generated by the hydrogen peroxide and the ferrous sulfate and can be used as a fluorescent probe of the hydroxyl radical. (2) According to the invention, after 20 mu L of hydrogen peroxide is added into the lycorine solution containing 1.176 mu M, the fluorescence intensity of the lycorine solution is not greatly changed along with the increase of the concentration of the added hydrogen peroxide, and the lycorine solution has no fluorescence response to the hydrogen peroxide and cannot be used as a fluorescence probe of the hydrogen peroxide. (3) According to the invention, after 0.1162mM hydroxyl radical is added into the solution containing 1.176 μ M lycorine, the fluorescence intensity of the lycorine at the maximum emission peak of 414nm begins to decrease, a new peak appears at the emission peak of 364nm, the maximum emission peak appears at 364nm when the concentration of the added hydroxyl radical is 0.3954mM, and the fluorescence intensity of the lycorine is enhanced by 8 times. When the lycorine is added with hydroxyl radical with very low concentration (0.1162mM), the lycorine can generate fluorescence response, and the fluorescence intensity of the lycorine has a quadratic function relation with the concentration of the added hydroxyl radical, which shows that the lycorine can be used as a fluorescent probe of the hydroxyl radical, the detection is very sensitive, and even micromolar hydroxyl radical can be accurately quantified.

The lycorine has good fluorescence response to hydroxyl free radicals, and the fluorescence intensity of the lycorine has close relation with the amount of the added hydroxyl free radicals, so that the micro-mole hydroxyl free radicals can be accurately quantified.

Drawings

The advantages and realisation of the invention will be more apparent from the following detailed description, given by way of example, with reference to the accompanying drawings, which are given for the purpose of illustration only, and which are not to be construed in any way as limiting the invention, and in which:

FIG. 1 is a structural diagram of lycorine

FIG. 2 is a graph showing the change of fluorescence spectrum of lycorine (1.176. mu.M) in the present invention after adding different ions (5.8. mu.M-56.49. mu.M), wherein a represents 1.176. mu.M of lycorine; b represents 5.8. mu.M potassium chloride; c represents 5.8. mu.M magnesium sulfate; d represents 5.7. mu.M calcium chloride; e represents 5.7. mu.M sodium hypochlorite; f represents 5.7. mu.M hydrogen peroxide; g represents 5.6. mu.M ferrous sulfate; h represents 56.49 μ M hydrogen peroxide; r represents 56.49 μ M ferrous sulfate

FIG. 3 is a graph showing the change of fluorescence spectrum of the mixture of lycorine (1.176. mu.M) and hydrogen peroxide (0-500 mM) at different concentrations, wherein None represents 1.176. mu.M of lycorine solution; 10. mu.M, 200. mu.M, 500. mu.M, 50mM, 80mM, 100mM, 500mM respectively indicate the addition of hydrogen peroxide at different concentrations.

FIG. 4 is a graph showing the change of fluorescence spectrum of lycorine (1.176. mu.M) in solution of 1.176. mu.M lycorine after 5 minutes addition of hydroxyl radicals (0-0.3954 mM) at different concentrations; 0.1162mM, 0.1449mM, 0.2017mM, 0.2579mM, 0.2857mM, 0.3409mM, 0.3954mM respectively represent the hydroxyl radical added at different concentrations.

FIG. 5 is a bar graph showing the change of fluorescence intensity of the present invention after adding different ions to lycorine (1.176. mu.M), wherein T0 indicates that OH, KCl, MgSO is not added to lycorine₄、CaCl₂、NaClO、H₂O₂、FeSO₄The fluorescence intensity at EX of 248nm and EM of 364 nm; t1 shows addition of 0.3683mM (. OH), 58. mu.M (KCl, MgSO)₄)、57μM(CaCl₂、NaClO、H₂O₂)、56μM(FeSO₄) The fluorescence intensity at an emission wavelength of 364 nm.

FIG. 6 is a graph showing the relationship between the second order function of the present invention at 364nm emission wavelength after adding hydroxyl radical at various concentrations to lycorine (1.176. mu.M) for 5 minutes.

Detailed Description

As shown in fig. 1 to fig. 6, the method for measuring lycorine as a fluorescent probe for detecting hydroxyl radicals of the present invention comprises the following steps:

(1) determining the excitation and emission wavelengths of lycorine: detecting with a fluorescence spectrophotometer, adding 3200 μ L of lycorine solution with concentration of 0.6623 μ M into a four-side light colorimetric dish, initially setting parameters, selecting an excitation wavelength of 360nm (initial setting, and then precisely selecting the excitation wavelength), setting the scanning wavelength range to be 380-640nm (the scanning initial wavelength should be 10-20nm longer than the excitation wavelength, and the scanning end wavelength should be 2 times shorter than the excitation wavelength to avoid the frequency doubling peak of the instrument), finding out that the maximum excitation wavelength of lycorine is 248nm and the maximum emission wavelength is 410nm,

(2) the detection of different ion fluorescence responses to lycorine: setting the determined excitation wavelength as a fixed value, starting scanning within a scanning range of 258-478 nm, wherein the obtained curve is the fluorescence curve of the original lycorine solution, sequentially adding 20 μ L of 10mM calcium chloride, potassium chloride, magnesium sulfate, sodium hypochlorite, hydrogen peroxide and ferrous sulfate solution into 1.176 μ M lycorine solution, scanning to obtain fluorescence response diagrams of different ions to the lycorine solution, storing data,

(3) and (3) detecting the fluorescence response of hydrogen peroxide with different concentrations to lycorine: adding 3400 μ L of lycorine solution with concentration of 1.176 μ M into a four-side light colorimetric cuvette, setting excitation wavelength at 248nm, scanning within 258-478 nm, scanning to obtain lycorine original solution curve, sequentially adding 20 μ L of 10 μ M, 200 μ M, 500 μ M, 50mM, 80mM, 100mM, and 500mM hydrogen peroxide solution into the colorimetric cuvette, scanning to obtain fluorescence response graphs of different concentrations to lycorine solution, storing the data obtained by scanning,

(4) and (3) detecting the fluorescence response of hydroxyl radicals with different concentrations to lycorine: hydroxyl radical is generated by the reaction of 3% hydrogen peroxide and ferrous sulfate, the lycorine raw solution is firstly scanned, 3400 mu L of 1.176 mu M lycorine solution with the concentration is added into a four-side light colorimetric dish, the excitation wavelength is set to be 248nm, the scanning range is set to be 258nm-478nm, the fluorescence curve of the lycorine raw solution is obtained, 20 mu L of 10mM hydrogen peroxide is firstly added, scanning is started, scanning is carried out again after 2-5 minutes after the scanning is finished until the fluorescence curve is stable and does not change any more (the error caused by the hydrogen peroxide is reduced), and the added hydrogen peroxide curve is deleted; then adding 20 mu L2.5mM ferrous sulfate solution, starting scanning, wherein the obtained curve is a fluorescence response graph of 0.1162mM hydroxyl free radicals to lycoris radiata alkali solution after 5s, scanning again after waiting for 5 minutes, and recording and storing the curve; continuously adding 20 mu L of 10mM hydrogen peroxide for scanning, observing the curve until the curve is not changed any more and then deleting the curve; and adding 20 mu L of 10mM ferrous sulfate solution, scanning to obtain a fluorescence response graph of 0.1449mM hydroxyl free radical to lycoris radiata alkali solution for 5s and 5min, not adding any more until the concentration of the added hydroxyl free radical is 0.3954mM, and recording and storing the obtained graphs and data of 5s and 5min for later data analysis.

Fluorescence detection of lycorine on different ions, as shown in FIG. 2, in order to verify the fluorescence response of different ions to lycorine, 20 μ L of KCl and MgSO 5.8 μ M was added₄5.7 μ M CaCl₂、NaClO、H₂O₂When the fluorescent substance was added to a solution containing 1.176. mu.M of lycorine, respectively, at 248nm as EX and at 414nm as EM, it was found from FIG. 2 that there was no change in the fluorescence intensity at 414nm, the maximum emission wavelength. When 20. mu.M FeSO of 5.6. mu.M concentration was added₄The fluorescence intensity was reduced because hydroxyl radicals were generated by the reaction of the added hydrogen peroxide with ferrous sulfate, the fluorescence intensity was not changed when 20. mu.L of hydrogen peroxide was continuously added at a concentration of 5.7. mu.M, and when 20. mu.L of FeSO was added at a concentration of 5.6. mu.M₄The fluorescence intensity begins to decrease again because lycorine has a fluorescent response to hydroxyl radicals generated by hydrogen peroxide and ferrous sulfate.

As can be seen from FIG. 2, 20. mu.L of KCl, MgSO 5. mu.M was added to a solution containing 1.176. mu.M lycorine₄5.7 μ M CaCl₂、NaClO、H₂O₂There was no change in fluorescence intensity when 20. mu.L of 5.6. mu.M FeSO was added₄While the fluorescence intensity at 414nm of the maximum emission peak was reduced, 20. mu.L of 5.7. mu.MH was added₂O₂The fluorescence intensity did not change until 20. mu.L of 5.6. mu.M FeSO was added₄The intensity of the fluorescence begins to decrease again, so that it can be concluded that 1.176 μ M lycorine responds to hydroxyl radical generated by hydrogen peroxide and ferrous sulfate and can be used as fluorescence of hydroxyl radicalAnd (3) a probe.

As shown in FIG. 3, in order to verify the fluorescent response of lycorine to hydrogen peroxide, 20 μ L of hydrogen peroxide with different concentrations was sequentially added to 1.176 μ M of lycorine, with 248nm as EX and EM as 414nm, and it was observed that the fluorescence intensity of lycorine decreased slightly from that without hydrogen peroxide with the addition of non-oxidizing hydrogen concentration, but the change was slight.

As can be seen from FIG. 3, after 20. mu.L of hydrogen peroxide is added to a solution containing 1.176. mu.M of lycorine, the fluorescence intensity of the lycorine solution does not change greatly with the increase of the concentration of the added hydrogen peroxide, and the lycorine solution has no fluorescence response to the hydrogen peroxide and cannot be used as a fluorescence probe of the hydrogen peroxide.

The fluorescence response of lycorine to different concentrations of hydroxyl radicals is shown in fig. 4-6.

As shown in FIG. 4, in lycorine (1.176. mu.M), with EX of 248nm and 411nm of EM, the maximum emission peak appeared at 414nm before the hydroxyl radical was not added, whereas when the hydroxyl radical concentration was 0.1162mM, a new peak appeared at 364nm of EM and the fluorescence intensity of the new emission peak was 100 nm.

As shown in FIG. 5, the fluorescence intensity of lycorine is increased with the increasing concentration of hydroxyl radical, and when the hydroxyl radical is added to 0.3954mM, the fluorescence intensity of the maximum emission peak at 364nm is increased by 8 times.

As shown in FIG. 6, the increase in fluorescence intensity of lycorine was strongly correlated with the concentration of hydroxyl radical added, and had a quadratic function between the concentrations of 0.1449mM and 0.3683 mM.

When 0.1162mM hydroxyl radical is added into 1.176. mu.M lycorine solution, the fluorescence intensity of lycorine at the maximum emission peak of 414nm begins to decrease, a new peak appears at the emission peak of 364nm, and when the hydroxyl radical is added at the concentration of 0.3954mM, the maximum emission peak appears at 364nm, and the fluorescence intensity of lycorine is enhanced by 8 times. When the lycorine is added with hydroxyl radical with very low concentration (0.1162mM), the lycorine can generate fluorescence response, and the fluorescence intensity of the lycorine has a quadratic function relation with the concentration of the added hydroxyl radical, which shows that the lycorine can be used as a fluorescent probe of the hydroxyl radical, the detection is very sensitive, and even micromolar hydroxyl radical can be accurately quantified.

The embodiments of the present invention have been described in detail, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention should be covered by the present patent.