Metal ion detection method based on three-color fluorescent carbon dot sensing array

Abstract

The invention discloses a metal ion detection method based on a three-color fluorescent carbon dot sensing array, and belongs to the technical field of ion detection. According to the method, glutathione, formamide, manganese perchlorate hexahydrate and zinc chloride are used as raw materials, naked carbon dots (CDs), manganese-doped carbon dots (Mn-CDs) and zinc-doped carbon dots (Zn-CDs) are obtained through a one-step solvothermal method, and three kinds of carbon dot solutions are mixed with different kinds of metal ion standard solutions respectively for fluorescence detection. A fluorescent sensing array and a database of a single metal ion solution and a composite metal ion solution are constructed, and qualitative and quantitative detection of a to-be-detected solution is realized. The array sensor constructed by the method disclosed by the invention has high sensitivity and high selectivity, can distinguish and identify as many as 10 metal ions, also realizes quantitative detection on 5 metal ions, and has the capability of accurately identifying and quantifying the metal ions in an actual sample.

Description

Technical Field

[0001] The invention belongs to the technical field of ion detection, and in particular relates to a metal ion detection method based on a three-color fluorescent carbon dot sensor array. Background Art

[0002] Heavy metal ions have attracted increasing attention due to their high toxicity, strong bioaccumulation and non-biodegradability. The development of fast, convenient and accurate methods for heavy metal ion detection is of great significance for environmental protection and pollution control. In recent years, people have constructed a class of array sensors based on cross-reaction receptors by simulating the mammalian olfactory system. Such sensors can produce specific response signals for different analytical components, which provides the possibility of rapid identification of the types of mixed metal ions in unknown samples with the assistance of statistical analysis methods. However, most of the fluorescent sensor elements currently developed for metal ion detection have problems such as high detection cost, complex synthesis or environmental pollution, which limits their application in practical detection. Carbon dots (CDs) are a kind of carbon-based nanomaterials with fluorescent properties. Because of their advantages such as adjustable emission, resistance to photobleaching, low cost and easy preparation, they have become ideal cross-reaction probes in sensor arrays.

[0003] At present, there are few reports on array sensors for detecting metal ions using carbon dots as sensing elements. The reported array sensors based on carbon dots only focus on qualitative identification of multiple targets and cannot achieve quantitative detection of metal ions. For example, Xu et al. used carbon dots and new lanthanide complexes to construct a simple multi-emission fluorescence sensor array, which can sensitively detect seven heavy metal ions in the range of 0.05-50μM and effectively identify binary mixed samples, but the array sensor cannot quantitatively analyze the concentration of heavy metal ions in the sample. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a metal ion detection method based on a three-color fluorescent carbon dot sensor array to solve the technical problem that the existing carbon dots have limited detection ability for metal ions and cannot perform quantitative detection.

[0005] To achieve the above object, the technical solution adopted by the present invention is: to provide a metal ion detection method based on a three-color fluorescent carbon dot sensor array, comprising the following steps:

[0006] S1. Mixing a peptide substance and an amide substance, and then reacting them at 150-170° C. for 7-9 hours to obtain a reaction solution; dialyzing the reaction solution for 6-7 days, and then filtering and freeze-drying the filtrate to obtain bare carbon dots (CDs); the ratio of the peptide substance to the amide substance is 100-200 mg: 10-20 mL;

[0007] S2, mixing a peptide substance, an amide substance and a metal salt, and then reacting at 150-170° C. for 7-9 hours to obtain a reaction solution; dialyzing the reaction solution for 6-7 days, and then filtering, and freeze-drying the filtrate to obtain metal-doped carbon dots; the ratio of the peptide substance, the amide substance and the metal salt is 100-200 mg: 10-20 mL: 1-3 mg;

[0008] S3, performing fluorescence detection on the metal-doped carbon dot solution and the bare carbon dot solution respectively to obtain the original fluorescence intensity F0, and then mixing the metal-doped carbon dot solution and the bare carbon dot solution with different types of metal ion standard solutions respectively to obtain the fluorescence intensity F of different types of metal ions; finally, obtaining the relative fluorescence intensity F′ by normalization, converting the relative fluorescence intensity F′ into a typical discriminant factor by linear discriminant analysis, constructing a fluorescence sensing array and database of a single metal ion solution and a composite metal ion solution, and obtaining a standard curve of metal ions;

[0009] S4. The solution to be tested is discriminated by linear discriminant analysis and hierarchical cluster analysis to obtain the type of metal ions in the solution to be tested, and the concentration of the metal ions in the solution to be tested is calculated by the standard curve of the corresponding metal ions.

[0010] On the basis of the above technical solution, the present invention can also be improved as follows:

[0011] Furthermore, the peptide substance is a small molecule peptide substance containing a thiol group, the amide substance is formamide, and the metal salt is a water-soluble manganese salt or a water-soluble zinc salt.

[0012] Furthermore, the peptide substance is glutathione, the manganese salt is manganese perchlorate hexahydrate, and the zinc salt is zinc chloride.

[0013] Each metal ion has its own unique inherent properties such as electronic structure, electronegativity, ionic radius and coordination ability. These properties determine the interaction mode and intensity between metal ions and carbon dots, thus affecting the overall performance of doped carbon dots. Different metal ions can change the electronic structure and band structure of carbon dots, and have different effects on the fluorescence of carbon dots, thus affecting their optical, electrical and catalytic properties. Metal ions may also act as active centers or catalytic sites to promote the activity and selectivity of carbon dots in specific reactions. Based on this, metal-doped carbon dots are synthesized by selecting manganese ions with moderate coordination ability and zinc ions with strong coordination ability as doping metals. The doping of zinc ions and manganese ions makes the coordination sites on the surface of carbon dots occupied to different degrees, so that the metal ions added later compete with them for coordination, thereby affecting the changes in the fluorescence of metal ions.

[0014] Furthermore, the metal-doped carbon dots are manganese-doped carbon dots (Mn-CDs) or zinc-doped carbon dots (Zn-CDs).

[0015] Furthermore, the pore size of the filter membrane used for filtration is 0.2-0.3 μM.

[0016] Furthermore, the drying temperature is -90 to -70°C, and the drying time is 70 to 74 hours.

[0017] Furthermore, the metal ion is Ag + , Cu 2+ , Fe 3+ , Hg 2+ , Yb 3+ 、Zn 2+ , Pb 2+ Cr 3+ 、Zr 4+ and Cd 2+ .

[0018] Furthermore, the calculation formula of the relative fluorescence intensity F′ is:

[0019]

[0020] The beneficial effects of the present invention are as follows: the method adopts a one-step solvothermal method to obtain the target product, the synthesis is simple and easy to operate, the cost is low, and the three carbon dots (CDs, Mn-CDs and Zn-CDs) obtained have unique fluorescence properties and are effective for Ag. + , Cu 2+ , Fe 3+ , Hg 2+ , Yb 3+ 、Zn 2+ , Pb 2+ Cr 3+ 、Zr 4+ and Cd 2+ The array sensor constructed by the method of the present invention has high sensitivity and high selectivity. Under the doping of zinc and manganese ions, the three carbon dots show different fluorescence responses for different metal ions, and can distinguish and identify up to 10 metal ions (Ag + , Cu 2+ , Fe 3+ , Hg 2+ , Yb 3+ 、Zn 2+ , Pb 2+ Cr 3 + 、Zr 4+ and Cd 2+ ), and also five metal ions (Zn 2+ 、Ag + , Hg 2+ Cr 3+ and Cd 2+) achieves quantitative detection and has the ability to accurately identify and quantify metal ions in actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The synthetic route of three carbon dots;

[0022] Figure 2 It is the detection process of the method of the present invention;

[0023] Figure 3 are the UV-visible absorption, excitation, and emission spectra of CDs;

[0024] Figure 4 are the UV-visible absorption, excitation and emission spectra of Mn-CDs;

[0025] Figure 5 are the UV-visible absorption, excitation and emission spectra of Zn-CDs;

[0026] Figure 6 Fluorescence spectra of CDs before and after the addition of 10 heavy metal ions;

[0027] Figure 7 Fluorescence spectra of Mn-CDs before and after the addition of 10 heavy metal ions;

[0028] Figure 8 Fluorescence spectra of Zn-CDs before and after the addition of 10 heavy metal ions;

[0029] Fig. 9 This is the LDA standard score graph when the final concentration of metal ions is 20 μM;

[0030] Fig.10 This is the LDA standard score graph when the final concentration of metal ions is 10 μM;

[0031] Fig.11 This is the LDA standard score graph when the final concentration of metal ions is 5 μM;

[0032] Fig.12 For the sensing array to Pb 2+ With Ag + LDA standard score plot for binary mixtures;

[0033] Fig.13 For the sensing array to Pb 2+ With Ag + HCA analysis of binary mixtures;

[0034] Fig.14 Zn 2+ The linear fitting curve between factor 1;

[0035] Fig.15Ag + The linear fitting curve between factor 1;

[0036] Fig.16 Cd 2+ The linear fitting curve between factor 1;

[0037] Fig.17 Hg 2+ The linear fitting curve between factor 1;

[0038] Fig.18 Cr 3+ The linear fitting curve between factor 1;

[0039] Fig.19 The test result of the sample to be tested. DETAILED DESCRIPTION

[0040] The specific embodiments of the present invention are described below to facilitate the understanding of the present invention by those skilled in the art. If the specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

[0041] Example 1

[0042] A method for preparing bare carbon dots (CDs), comprising the following steps: mixing 0.7 g of glutathione and 70 mL of formamide, and then reacting at 150° C. for 8 h to obtain a dark green reaction solution; transferring the reaction solution to a 3500 KD dialysis bag for 6 days, changing water 4 times a day, and then filtering with a filter membrane with a pore size of 0.22 μM, and vacuum drying the filtrate at -75° C. for 73 h to obtain bare carbon dots (CDs).

[0043] A method for preparing manganese-doped carbon dots (Mn-CDs) comprises the following steps: mixing 200 mg of glutathione, 20 mL of formamide and 3 mg of manganese perchlorate hexahydrate, and then reacting at 170° C. for 7 hours to obtain a yellow-green reaction solution; transferring the reaction solution to a 3500 KD dialysis bag for dialysis for 7 days, and then filtering with a filter membrane with a pore size of 0.3 μM, and vacuum drying the filtrate at -90° C. for 70 hours to obtain manganese-doped carbon dots.

[0044] A method for preparing zinc-doped carbon dots (Zn-CDs), comprising the following steps: mixing 100 mg of glutathione, 10 mL of formamide and 1 mg of zinc chloride, and then reacting at 150° C. for 9 hours to obtain an emerald green reaction solution; transferring the reaction solution to a 3500KD dialysis bag for 6 days, and then filtering with a filter membrane with a pore size of 0.2 μM, and vacuum drying the filtrate at -70° C. for 74 hours to obtain zinc-doped carbon dots.

[0045] A method for detecting metal ions based on a three-color fluorescent carbon dot sensor array comprises the following steps:

[0046] S1. The bare carbon dots, manganese-doped carbon dots and zinc-doped carbon dots solutions were subjected to fluorescence detection respectively to obtain the original fluorescence intensity F0, and then the bare carbon dots, manganese-doped carbon dots and zinc-doped carbon dots solutions were mixed with different types of metal ion standard solutions respectively to obtain the fluorescence intensity F of different types of metal ions; finally, the relative fluorescence intensity F′ was obtained by normalization, and the relative fluorescence intensity F′ was converted into a typical discriminant factor by linear discriminant analysis, and the fluorescence sensing array and database of single metal ion solution and composite metal ion solution were constructed to obtain the standard curve of metal ions; the calculation formula of the relative fluorescence intensity F′ is:

[0047]

[0048] S2. The solution to be tested is discriminated by linear discriminant analysis and hierarchical cluster analysis to obtain the type of metal ions in the solution to be tested, and the concentration of the metal ions in the solution to be tested is calculated by the standard curve of the corresponding metal ions.

[0049] Example 2

[0050] A method for preparing bare carbon dots (CDs), the synthesis flow chart is as follows Figure 1 As shown, the process comprises the following steps: mixing 0.7 g of glutathione and 70 mL of formamide, and then reacting at 160° C. for 8 h to obtain a dark green reaction solution; transferring the reaction solution to a 3500 KD dialysis bag and dialyzing it for 6 days, changing the water 4 times a day, and then filtering it with a filter membrane with a pore size of 0.22 μM, and placing the filtrate at -80° C. and vacuum drying it for 72 h to obtain bare carbon dots (CDs).

[0051] A manganese-doped carbon dot (Mn-CDs), whose preparation method is different from the preparation method of the bare carbon dots (CDs) in this embodiment in that: a raw material manganese perchlorate hexahydrate is added, that is, 0.7 g glutathione, 70 mL formamide and 10 mg manganese perchlorate hexahydrate are mixed, and the rest of the preparation process is the same as the preparation process of the bare carbon dots to obtain manganese-doped carbon dots (Mn-CDs).

[0052] A zinc-doped carbon dot (Zn-CDs), whose preparation method is different from the preparation method of the bare carbon dots (CDs) in this embodiment in that: adding a raw material zinc chloride, that is, mixing 0.7g glutathione, 70mL formamide and 10mg zinc chloride, and the rest of the preparation process is the same as the preparation process of the bare carbon dots, to obtain zinc-doped carbon dots (Zn-CDs).

[0053] The optical properties of bare carbon dots (CDs), manganese-doped carbon dots (Mn-CDs) and zinc-doped carbon dots (Zn-CDs) were investigated. Figure 3-Figure 5 As shown, the inset is a photo of the carbon dot solution under visible light (left) and 405nm ultraviolet light (right). CDs, Mn-CDs and Zn-CDs solutions show red, light red and bright red fluorescence under ultraviolet light, respectively. In the fluorescence spectrum, it can be seen that the maximum excitation wavelength of CDs is 420nm, and there are emissions at 658nm and 687nm; the optimal excitation wavelength of Mn-CDs is at 420nm, with emission peaks at 658nm and 687nm respectively; Zn-CDs has emission and excitation characteristic peaks at 658nm and 420nm. The three carbon dots absorb at 420nm and 640nm, but the absorption peak intensities are quite different.

[0054] A metal ion detection method based on a three-color fluorescent carbon dot sensor array, such as Figure 2 As shown, the following steps are included:

[0055] S1, the bare carbon dots, manganese-doped carbon dots and zinc-doped carbon dots solutions were subjected to fluorescence detection respectively to obtain the original fluorescence intensity F0, and then the bare carbon dots, manganese-doped carbon dots and zinc-doped carbon dots solutions were mixed with different types of metal ions (Ag + , Cu 2+ , Fe 3+ , Hg 2+ , Yb 3+ 、Zn 2+ , Pb 2+ Cr 3+ 、Zr 4+ and Cd 2+ ) standard solution, the final concentration of metal ions was 10 μM, and the final concentration of CDs was 10 μg / mL, and the fluorescence intensity F of different types of metal ions was obtained; finally, the relative fluorescence intensity F' was obtained by normalization, and the relative fluorescence intensity F' was converted into a typical discriminant factor by linear discriminant analysis, and the fluorescence sensing array and database of single metal ion solution and composite metal ion solution were constructed to obtain the standard curve of metal ions; the calculation formula of relative fluorescence intensity F' is:

[0056] The recording process of fluorescence spectrum is as follows: the carbon dot solution and the metal ion standard solution are mixed and incubated for 10 minutes, and then the solution is detected and the fluorescence spectrum is recorded using a fluorescence spectrometer, with the excitation light at 420nm, the voltage at 400V, and the excitation and emission slits at 5.0nm. The recording process of fluorescence response is as follows: the carbon dot solution and the metal ion standard solution are mixed and incubated for 10 minutes, and then the fluorescence intensity F of the solution at 687nm is recorded using an ELISA reader, and each group is repeated 5 times.

[0057] S2. The solution to be tested is discriminated by linear discriminant analysis and hierarchical cluster analysis to obtain the type of metal ions in the solution to be tested, and the concentration of the metal ions in the solution to be tested is calculated by the standard curve of the corresponding metal ions.

[0058] Depend on Figure 6-Figure 8 It can be observed that the fluorescence spectra of the three carbon dots have changed to varying degrees. 2+ can quench the fluorescence of the three CDs to a great extent, while Ag + With Cu 2+ Zn 2+ The quenching ability of Zn is slightly weaker, but it can effectively quench the fluorescence of CDs and Mn-CDs, while Zn 2+ It can greatly enhance the emission peak intensity of CDs and Mn-CDs at 658nm, but has little effect on Zn-CDs; this proves the feasibility of using these three types of carbon dots as sensing probes for heavy metal ion identification.

[0059] The bare carbon dots (CDs), manganese-doped carbon dots (Mn-CDs) and zinc-doped carbon dots (Zn-CDs) used in the following experiments are the three-color fluorescent carbon dots prepared in Example 2.

[0060] Experimental Example 1 Identification and Quantification Capabilities of Array Sensors

[0061] 1. Qualitative identification

[0062] In order to verify the recognition ability of the array sensor constructed by the present invention, different types of metal ion solutions (Ag + , Cu 2+ , Fe 3+ , Hg 2+ , Yb 3+ 、Zn 2+ , Pb 2+ Cr 3+ 、Zr 4+ and Cd 2+, final concentrations of 5, 10 and 20 μM, respectively) were mixed with 100 μL of three fluorescent probes (CDs, Mn-CDs and Zn-CDs, all at 10 μg / mL) to form a sensor array. After incubation at room temperature for 10 min, the fluorescence spectrum of the mixture was recorded, and the fluorescence intensity was used to distinguish various metal ions. Each experimental group was repeated 5 times to obtain a training data matrix of 3 carbon dots × 10 metal ions × 5 replicates. Figure 9-11 As shown, when the final concentration of metal ions was 5, 10 and 20 μM, the array sensor had good recognition ability, and all 10 heavy metal ions detected were correctly identified, with a recognition accuracy of 100%.

[0063] In order to ensure the recognition ability of mixed metal ions, the mixed heavy metal ions were measured by using array sensing. The specific steps are: + and Pb 2+ Binary mixture (Ag + :Pb 2+ =9:1, 7:3, 5:5, 3:7 and 1:9, with a total metal ion concentration of 10 μM) were mixed with 100 μL of each of the three fluorescent probes (CDs, Mn-CDs and Zn-CDs, all at a concentration of 10 μg / mL), and their fluorescence responses were recorded 5 times, and the mixture was identified. Fig.12 As shown, pure Ag can be identified in the LDA graph. + and Pb 2+ and mixtures thereof, and arranged in exact order of molar ratio on Factor1. Fig.13 As shown, the HCA results also showed that each of the 35 samples was accurately classified into the corresponding category, which indicated that the array sensor exhibited good resolution for mixed metal ions of different concentrations and that the CDs-based sensor array could be used to identify mixtures containing mixed metal ions.

[0064] 2. Quantitative detection

[0065] In order to determine the metal ion (Zn 2+ 、Ag + , Hg 2+ Cr 3+ and Cd 2+ ) sensitivity, using the values ​​of factor 1 in the typical scoring diagram to relate a range of Zn concentrations 2+ (0.25-2.5μM), Ag + (1-2.5μM), Hg 2+ (0.05-1000μM), Cr 3+ (0.01-0.25μM) and Cd 2+(0.05-2.5 μM), it was verified that there was a good linear relationship between factor 1 and metal ion concentration, such as Figure 14-18 As shown, it shows that the fluorescent sensor array can also be used to quantitatively detect specific heavy metal ions. Subsequently, a database containing 5 metal ions was constructed to quantitatively analyze heavy metal ions. Each curve starts at 0 μM and is connected to the data points representing the specific heavy metal ions at the corresponding concentration, showing the continuous spatial change of a single heavy metal ion with increasing concentration. The results of quantitative detection of metal ions by factor 1 scoring are shown in Table 1.

[0066] Table 1 Results of quantitative detection of metal ions by factor 1 scoring

[0067]

[0068]

[0069] Result analysis: As can be seen from Table 1, the method of the present invention can be used to qualitatively identify and quantitatively detect the content of specific heavy metal ions, with a detection limit as low as 11.46 nM, which can achieve accurate identification and detection of heavy metal ions.

[0070] Experimental Example 3: Actual Sample Testing

[0071] The diluted tap water sample (tap water: deionized water = 1:10, v:v) and the lake water sample (lake water: deionized water = 1:20, v:v) were labeled as sample 1 and sample 2, respectively. At the same time, four samples with two different concentrations of metal ions were prepared and labeled as samples 3-6, respectively. Then, the fluorescence signals of samples 1-6 were subjected to ICP detection and LDA analysis. The ICP results are shown in Tables 2 and 3, and the LDA analysis is shown in Tables 2 and 3. Fig.19 As shown, samples 1 and 2 are classified as Zn 2+ , samples 3 and 4 were classified as Cd 2+ , samples 5 and 6 were classified as Hg 2+ Finally, according to Figure 14-18 The linear equation of the present invention was used to quantitatively detect samples 1-6, which was roughly consistent with the ICP results, with a relative deviation between 3.69% and 16.82%, indicating that the present invention has the ability to accurately identify and quantify metal ions in actual samples.

[0072] Table 2 Test results of actual samples

[0073]

[0074] Table 3 Test results of spiked samples

[0075]

Claims

1. A metal ion detection method based on a three-color fluorescent carbon dot sensor array, characterized in that: The following steps are involved: S1. Mixing a peptide substance and an amide substance, and then reacting them at 150-170° C. for 7-9 hours to obtain a reaction solution; dialyzing the reaction solution for 6-7 days, and then filtering and freeze-drying the filtrate to obtain bare carbon dots; the ratio of the peptide substance to the amide substance is 100-200 mg: 10-20 mL; S2, mixing a peptide substance, an amide substance and a metal salt, and then reacting at 150-170° C. for 7-9 hours to obtain a reaction solution; dialyzing the reaction solution for 6-7 days, and then filtering, and freeze-drying the filtrate to obtain metal-doped carbon dots; the ratio of the peptide substance, the amide substance and the metal salt is 100-200 mg: 10-20 mL: 1-3 mg; S3, performing fluorescence detection on the metal-doped carbon dot solution and the bare carbon dot solution respectively to obtain the original fluorescence intensity F0, and then mixing the metal-doped carbon dot solution and the bare carbon dot solution with different types of metal ion standard solutions respectively to obtain the fluorescence intensity F of different types of metal ions; finally, obtaining the relative fluorescence intensity F′ by normalization, converting the relative fluorescence intensity F′ into a typical discriminant factor by linear discriminant analysis, constructing a fluorescence sensing array and database of a single metal ion solution and a composite metal ion solution, and obtaining a standard curve of metal ions; S4. The solution to be tested is discriminated by linear discriminant analysis and hierarchical cluster analysis to obtain the type of metal ions in the solution to be tested, and the concentration of the metal ions in the solution to be tested is calculated by the standard curve of the corresponding metal ions.

2. The metal ion detection method based on the three-color fluorescent carbon dot sensor array according to claim 1, characterized in that: The peptide substance is a small molecule peptide substance containing a thiol group, the amide substance is formamide, and the metal salt is a water-soluble manganese salt or a water-soluble zinc salt.

3. The metal ion detection method based on the three-color fluorescent carbon dot sensor array according to claim 2 is characterized in that: The peptide substance is glutathione, the manganese salt is manganese perchlorate hexahydrate, and the zinc salt is zinc chloride.

4. The metal ion detection method based on the three-color fluorescent carbon dot sensor array according to claim 1, characterized in that: The metal-doped carbon dots are manganese-doped carbon dots or zinc-doped carbon dots.

5. The metal ion detection method based on the three-color fluorescent carbon dot sensor array according to claim 1, characterized in that: The pore size of the filter membrane used for the filtration is 0.2-0.3 μM.

6. The metal ion detection method based on the three-color fluorescent carbon dot sensor array according to claim 1, characterized in that: The drying temperature is -90 to -70°C, and the drying time is 70 to 74 hours.

7. The metal ion detection method based on the three-color fluorescent carbon dot sensor array according to claim 1, characterized in that: The metal ion is Ag + , Cu 2+ , Fe 3+ , Hg 2+ , Yb 3+ 、Zn 2+ , Pb 2+ Cr 3+ 、Zr 4+ and Cd 2+ .

8. The metal ion detection method based on the three-color fluorescent carbon dot sensor array according to claim 1, characterized in that: The calculation formula of the relative fluorescence intensity F' is:

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