CN108318465B - A kind of detection method of heme solution concentration - Google Patents

Abstract

The invention provides a method for detecting the concentration of heme solution, which belongs to the field of biological detection. The invention uses thiamine instead of luminol, has low cost and is relatively environmentally friendly, the fluorescent signal is relatively stable, and the reproducibility of the detection result is improved. It has no fluorescence itself. In the presence of superoxide radicals, it oxidizes to generate sulfur pigments with fluorescent properties. The heme concentration is detected by fluorescence spectroscopy, which has better reproducibility than chemiluminescence detection. The data of the examples show that the detection method provided by the present invention has carried out 6 repeated determinations of 300 nmol/L heme, and the relative standard deviation is 6.8%.

Description

A kind of detection method of heme solution concentration

technical field

The invention relates to the technical field of biological detection, in particular to a method for detecting the concentration of heme solution.

Background technique

Heme, a natural iron porphyrin compound, plays a key role in oxygen transport, which enables the normal functioning of human organs. Excessive heme may lead to permanent secondary brain damage after hemorrhagic stroke, while heme deficiency may induce neuroglobulin expression in nerve cells, accelerating endogenous carbon monoxide production. In addition, as a new type of iron supplement, it is widely used in medicine, food and other fields.

At present, the reported methods for the detection of heme include chemiluminescence, electrochemical, capillary electrophoresis and fluorescence spectroscopy, etc., but there is a problem of poor reproducibility of the detection methods. For example, Xu et al. used the artemisinin-luminol system to achieve The chemiluminescence detection of heme was carried out by adding different concentrations of heme into the buffer solution containing luminol and artemisinin for chemiluminescence detection (see "Artemisinin-luminol chemiluminescence for forensic bloodstain detection using a smart phone as a detector"). ", G.B.Xu et al., Anal.Chem., 2017, 89: 6160-6165), but the luminol luminescence time is limited, the sample needs to be measured immediately after preparation, and the reproducibility is poor.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for detecting the concentration of heme solution with good reproducibility.

The invention provides a method for detecting the concentration of heme solution, comprising the following steps:

(1) Mix the heme solution to be tested, disodium EDTA, artemisinin, thiamine, and inorganic base with water, incubate the obtained mixed solution to be tested to obtain the incubation solution to be tested, and detect the the fluorescence intensity of the incubation solution to be tested;

(2) Obtain the concentration of heme in the heme solution to be measured according to the fluorescence intensity of the incubation solution to be measured and the predetermined standard curve obtained in the step (1); the standard curve takes the concentration of the standard heme solution as the horizontal Coordinate, the difference between the fluorescence intensity after adding the heme standard solution and the initial fluorescence intensity of the mixed solution of artemisinin and thiamine is the ordinate.

Preferably, the incubation temperature in the step (1) is 15-40°C, and the incubation time is 5-30 min.

Preferably, in the step (1), the molar ratio of artemisinin and thiamine in the heme solution to be tested is 10-2000:0.5-500.

Preferably, in the step (1), the inorganic base is sodium hydroxide or potassium hydroxide.

Preferably, in the step (1), the inorganic alkali is added in the form of an inorganic alkali solution, and the concentration of the inorganic alkali solution is 5-500 mmol/L.

Preferably, in the step (1), artemisinin is added in the form of an artemisinin solution, and the concentration of the artemisinin solution is 0.05-50 mmol/L.

Preferably, in the step (1), thiamine is added in the form of a thiamine solution, and the concentration of the thiamine solution is 5 μmol/L˜2 mmol/L.

Preferably, the linear range of the standard curve in the step (2) is 2.0-300.0 nmol/L.

The invention provides a method for detecting the concentration of heme solution, comprising the following steps: mixing the heme solution to be measured, disodium EDTA, artemisinin, thiamine, inorganic base and water, The mixed solution to be tested is incubated to obtain the incubation solution to be tested, and the fluorescence intensity of the incubation solution to be tested is detected; according to the fluorescence intensity of the incubation solution to be tested and a predetermined standard curve, the concentration of heme in the heme solution to be tested is obtained; The standard curve takes the concentration of the heme standard solution as the abscissa, and the difference between the fluorescence intensity after adding the heme standard solution and the initial fluorescence intensity of the mixed solution of artemisinin and thiamine as the ordinate. The invention uses thiamine instead of luminol, has low cost and is relatively environmentally friendly, the fluorescent signal is relatively stable, and the reproducibility of the detection result is improved. It has no fluorescence itself. In the presence of superoxide radicals, it oxidizes to generate sulfur pigments with fluorescent properties. The heme concentration is detected by fluorescence spectroscopy, which has better reproducibility than chemiluminescence detection. The data of the examples show that the detection method provided by the present invention has carried out 6 repeated determinations on the heme solution of 300 nmol/L, and the relative standard deviation is 6.8%. and has good selectivity.

Description of drawings

Fig. 1 is the change of fluorescence intensity after adding different concentrations of heme standard solution in the embodiment of the present invention 1;

Fig. 2 is the relation curve of fluorescence intensity and heme concentration of the system in Example 1 of the present invention, and the inset is the standard equation of fluorescence intensity change and heme;

Fig. 3 is the fluorescence intensity change of the system after the addition of other interfering substances in Example 2 of the present invention.

Detailed ways

The invention provides a method for detecting the concentration of heme solution, comprising the following steps:

(1) Mix the heme solution to be tested, disodium EDTA, artemisinin, thiamine, and inorganic base with water, incubate the obtained mixed solution to be tested to obtain the incubation solution to be tested, and detect the the fluorescence intensity of the incubation solution to be tested;

(2) Obtain the concentration of heme in the heme solution to be measured according to the fluorescence intensity of the incubation solution to be measured and the predetermined standard curve obtained in the step (1); the standard curve takes the concentration of the standard heme solution as the horizontal Coordinate, the difference between the fluorescence intensity after adding the heme standard solution and the initial fluorescence intensity of the mixed solution of artemisinin and thiamine is the ordinate.

In the present invention, the heme solution to be tested, disodium EDTA, artemisinin, thiamine and inorganic base are mixed with water to obtain the mixed solution to be tested and incubated to obtain the incubation solution to be tested, and the incubation solution to be tested is detected fluorescence intensity. In the present invention, the incubation temperature is preferably 15-40°C, more preferably 25°C, and the incubation time is preferably 5-30 min, more preferably 20 min.

In the present invention, the molar ratio of artemisinin to thiamine in the heme solution to be tested is preferably 10-2000:0.5-500.

In the present invention, the detection principle of the concentration of heme solution is as follows: artemisinin is decomposed to generate superoxide radicals in the presence of heme; thiamine itself has no fluorescence, and in the presence of superoxide radicals, it oxidizes to generate fluorescent sulfur Pigment, as shown in the following formula:

。

.

In the present invention, the pH value of the mixed solution to be tested is preferably 12-13. The invention adjusts the pH value of the mixed solution to be tested by the amount of the inorganic base. In the present invention, the inorganic base is preferably sodium hydroxide or potassium hydroxide. In the present invention, the inorganic alkali is preferably added in the form of an inorganic alkali solution, and the concentration of the inorganic alkali solution is preferably 5-500 mmol/L, more preferably 5, 10, 20, 50, 100, 200, 500 mmol/L. L; the volume of the inorganic base solution is 870 μL.

In the present invention, the concentration of the heme solution to be tested is preferably 2.0-300.0 nmol/L.

Metal ions, glucose and amino acids have no influence on the method for detecting the concentration of heme solution provided by the present invention, and will not affect the selectivity of the detecting method of the present invention. In the present invention, the metal ions preferably include cobalt ion, magnesium ion, copper ion, zinc ion and iron ion; the amino acid preferably includes phenylalanine, leucine, glycine, alanine and cysteine .

In the present invention, the artemisinin is preferably added in the form of an artemisinin solution, and the preferred concentration of the artemisinin solution is 0.05-50 mmol/L, more preferably 5 mmol/L; The addition volume is preferably 10 μL.

In the present invention, the thiamine is preferably added in the form of a thiamine solution, and the concentration of the thiamine solution is preferably 5 μmol/L to 2 mmol/L, more preferably 1 mmol/L; The added volume of the solution is preferably 100 μL.

The present invention has no special limitation on the amount of disodium ethylenediaminetetraacetate (EDTA), as long as it has no effect on the mixed solution to be tested.

The present invention has no special limitation on the addition order of the heme solution to be tested, disodium EDTA, artemisinin, thiamine, inorganic base and water, and the order of addition well known to those skilled in the art can be used. Specifically, disodium EDTA, artemisinin solution, thiamine solution, and heme solution to be tested are sequentially added to the inorganic alkali solution.

The present invention has no particular limitation on the method of detecting the fluorescence intensity of the incubation solution to be tested, and the method of measuring heme by fluorescence spectroscopy well known to those skilled in the art may be used.

After obtaining the fluorescence intensity value of the incubation solution to be measured, the present invention obtains the concentration of heme in the heme solution to be measured according to the obtained fluorescence intensity of the incubation solution to be measured and a predetermined standard curve; the standard curve is based on the standard heme solution. The concentration (c) of α is the abscissa, and the difference between the fluorescence intensity after adding the heme standard solution and the initial fluorescence intensity of the mixed solution of artemisinin and thiamine is the ordinate. In the embodiment of the present invention, the standard curve is ΔF=32.01+23.54c (the unit of c is nmol/L), the linear coefficient is 0.9954, the linear range is 2.0-300.0 nmol/L, and the detection limit is 0.68 nmol/L L. In the present invention, the method for obtaining the standard curve preferably includes the following steps:

Provide heme standard solution;

Mix disodium EDTA, artemisinin, thiamine and inorganic base with water, then add the heme standard solution, and detect the fluorescence intensity after adding the heme standard solution;

Taking the difference between the fluorescence intensity after adding the heme standard solution and the initial fluorescence intensity of the mixed solution of artemisinin and thiamine as the ordinate, and the concentration of the heme standard solution as the abscissa, the standard curve was obtained by linear fitting.

In the invention, the standard heme is mixed with water to obtain the heme standard solution, and the fluorescence intensity of the heme standard solution is measured. In the present invention, the concentration of the heme standard solution is preferably 0.002, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.5, 0.8, 2.0, 5.0 or 10.0 μmol/L. The source of the standard heme is not particularly limited in the present invention, and a standard heme known to those skilled in the art can be used.

After obtaining the heme standard solution, the present invention will mix disodium EDTA, artemisinin, thiamine, inorganic base with water, then add the heme standard solution, and detect adding the heme standard solution The difference between the fluorescence intensity after adding the heme standard solution and the initial fluorescence intensity of the mixed solution of artemisinin and thiamine is the ordinate, the concentration of the heme standard solution is the abscissa, and the linear fitting is obtained. standard curve line.

In the present invention, the molar ratio of artemisinin and thiamine is preferably consistent with the requirements defined in the above scheme, which will not be repeated here. The limitations of the present invention on the detection methods of artemisinin, thiamine, inorganic base, EDTA, and fluorescence intensity are consistent with the above requirements, and will not be repeated here.

The conditions for the incubation in the present invention are preferably the same as those described in the above scheme, which will not be repeated here.

Obtain the fluorescence intensity (F) after adding the heme standard solution, as the difference (ΔF) between the fluorescence intensity (F) after adding the heme standard solution and the initial fluorescence intensity (F ₀ ) of the mixed solution of artemisinin and thiamine ) is the dependent variable, the standard equation is fitted with the concentration (c) of the heme standard solution as the independent variable, and the concentration of heme in the heme solution to be tested is calculated according to the standard equation.

In order to further illustrate the present invention, the method for detecting the concentration of heme solution provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

10μL EDTA (0.1mol/L), 10μL artemisinin (5mmol/L), 10μL heme standard solutions of different concentrations (0.002, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.5, 0.8, 2.0, 5.0, 10.0 μmol/L) and 100 μL thiamine (1 mmol/L) were sequentially added to 880 μL sodium hydroxide solution (0.1 mol/L), and the fluorescence spectrum was measured after incubation at 25°C for 20 minutes. The linear relationship between the change value (ΔF) of the fluorescence intensity (ΔF) and the heme concentration of ₀ ) and after (F) and the concentration of heme was fitted to a standard curve, as shown in Figures 1-2, Figure 1 shows the fluorescence after adding different concentrations of heme standard solutions The change of intensity; the concentration of heme standard solution from bottom to top is 0, 0.002, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.5, 0.8, 2.0, 5.0, 10.0 μmol/L at one time, Figure 2 shows the fluorescence of the system The relationship between intensity and heme concentration, the inset is the standard equation of fluorescence intensity change and heme, the obtained standard equation is ΔF=32.01+23.54c (c is the concentration of heme, the unit is nmol/L), the linear coefficient is 0.9954, The linear range is 2.0～300.0nmol/L, and the detection limit is 0.68nmol/L.

Mix the heme solution to be tested, disodium EDTA, artemisinin, thiamine, and inorganic base with water to obtain the mixed solution to be tested and incubate, and the incubation conditions are the same as the above incubation conditions to obtain the incubation solution to be tested , detect the fluorescence intensity of the incubation solution to be tested, and calculate the concentration of heme in the heme solution to be tested according to the standard equation. The 300 nmol/L heme solution was repeatedly measured for 6 times, and the relative standard deviation was 6.8%.

Example 2

The steps are the same as those in Example 1, except that the heme solution to be tested is replaced with a solution containing cobalt ions, magnesium ions, copper ions, zinc ions and iron ions, glucose, phenylalanine, leucine, glycine, and alanine. Acid and cysteine, the heme solution to be tested was measured by fluorescence spectrum, and the results are shown in Figure 3. It can be seen from Figure 3 that the fluorescence intensity of the system did not change significantly after the addition of these substances, and only the system after the addition of heme The fluorescence intensity is obviously enhanced, indicating that the detection method of the present invention exhibits good selectivity and is not disturbed by some common metal ions and biomolecules.

The above descriptions are only preferred embodiments of the present invention, and do not limit the present invention in any form. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (8)

1. a detection method of heme solution concentration, comprises the following steps: (1) Mix the heme solution to be tested, disodium EDTA, artemisinin, thiamine, and inorganic base with water, incubate the obtained mixed solution to be tested to obtain the incubation solution to be tested, and detect the the fluorescence intensity of the incubation solution to be tested; (2) Obtain the concentration of heme in the heme solution to be measured according to the fluorescence intensity of the incubation solution to be measured and the predetermined standard curve obtained in the step (1); the standard curve takes the concentration of the standard heme solution as the horizontal Coordinate, the difference between the fluorescence intensity after adding the heme standard solution and the initial fluorescence intensity of the mixed solution of artemisinin and thiamine is the ordinate. 2 . The detection method according to claim 1 , wherein the incubation temperature in the step (1) is 15-40° C., and the incubation time is 5-30 min. 3 . 3 . The detection method according to claim 1 , wherein the molar ratio of artemisinin and thiamine in the mixed solution to be tested in the step (1) is 10-2000:0.5-500. 4 . 4. detection method according to claim 1, is characterized in that, in described step (1), inorganic base is sodium hydroxide or potassium hydroxide. 5. The detection method according to claim 1 or 4, wherein the inorganic base is added in the form of an inorganic base solution in the step (1), and the concentration of the inorganic base solution is 5-500 mmol/L. 6 . The detection method according to claim 1 , wherein, in the step (1), artemisinin is added in the form of an artemisinin solution, and the concentration of the artemisinin solution is 0.05-50 mmol/L. 7 . 7. detection method according to claim 1 is characterized in that, in described step (1), thiamine is added in the form of thiamine solution, and the concentration of described thiamine solution is 5 μmol/L～2mmol/ L. 8 . The detection method according to claim 1 , wherein the linear range of the standard curve in the step (2) is 2.0-300.0 nmol/L. 9 .

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