A method for detecting metal ions based on three kinds of fluorescent carbon dot sensing array

Abstract

The application discloses a kind of metal ion detection methods based on three kinds of fluorescent carbon dot sensing array, belong to ion detection technical field.The present application with glutathione, formamide, manganese perchlorate hexahydrate and zinc chloride as raw material, after one-step solvothermal method obtains naked carbon dot (CDs), manganese-doped carbon dot (Mn-CDs) and zinc-doped carbon dot (Zn-CDs), three kinds of carbon dot solution are mixed with different kinds of metal ion standard solution respectively and fluorescence detection is carried out, the fluorescence sensing array and database of single metal ion solution and composite metal ion solution are constructed, qualitative and quantitative detection to be measured solution is realized.The array sensing constructed by the method of the present application has high sensitivity and high selectivity, can distinguish and identify up to 10 kinds of metal ions, and also realizes quantitative detection to 5 kinds of metal ions among them, has the ability of accurately identifying and quantifying metal ions in actual sample.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of ion detection, and particularly relates to a metal ion detection method based on a three-fluorescent carbon dot sensing array. BACKGROUND

[0002] Heavy metal ions are increasingly concerned due to their high toxicity, strong biological accumulation and non-biodegradability. It is of great significance to develop a rapid, convenient and accurate heavy metal ion detection method for environmental protection and pollution control. In recent years, people have constructed a class of array sensors based on cross-reactive receptors by simulating the mammalian olfactory system. This kind of sensor can produce specific response signals for different analysis components, and provides the possibility for rapid identification of the type of mixed metal ions in unknown samples with the aid of statistical analysis methods. However, most of the fluorescent sensing elements developed for metal ion detection have problems such as high detection cost, complex synthesis or environmental pollution, which limits their application in actual detection. Carbon dots (CDs) are a kind of carbon-based nanomaterials with fluorescent properties. Because of their adjustable emission, light bleaching resistance, low cost and easy preparation, they have become ideal cross-reactive probes in sensor arrays.

[0003] Currently, 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 the qualitative identification of multiple targets and cannot achieve quantitative detection of metal ions. For example, Xu et al. used carbon dots and a novel lanthanide complex to construct a simple multi-emission fluorescent sensor array. The sensor array can sensitively detect seven heavy metal ions in the range of 0.05-50 μM, and can effectively identify binary mixed samples, but the array sensor cannot quantitatively analyze the concentration of heavy metal ions in the sample. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a metal ion detection method based on a three-fluorescent carbon dot sensing array, so as to solve the technical problems that the existing carbon dots have limited detection ability for metal ions and cannot quantitatively detect.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is to provide a metal ion detection method based on a three-fluorescent carbon dot sensing array, comprising the following steps:

[0006] S1, mixing a peptide substance and an amide substance, and then reacting at 150-170℃ for 7-9h to obtain a reaction liquid; dialyzing the reaction liquid for 6-7 days, and then filtering, freezing and drying the filtrate to obtain naked carbon dots (CDs); the ratio of the peptide substance to the amide substance is 100-200mg:10-20mL;

[0007] S2. Mix peptides, amides, and metal salts, and then react at 150-170°C for 7-9 hours to obtain a reaction solution; dialyze the reaction solution for 6-7 days, then filter, and freeze-dry the filtrate to obtain metal-doped carbon dots; the ratio of peptides, amides, and metal salts is 100-200 mg: 10-20 mL: 1-3 mg;

[0008] S3. Perform fluorescence detection on the metal-doped carbon dot solution and the bare carbon dot solution separately to obtain the original fluorescence intensity F0. Then, mix the metal-doped carbon dot solution and the bare carbon dot solution with standard solutions of different types of metal ions to obtain the fluorescence intensity F of different types of metal ions. Finally, normalize to obtain the relative fluorescence intensity. Linear discriminant analysis was used to determine the relative fluorescence intensity. The results were transformed into typical discriminant factors, and fluorescence sensing arrays and databases for single metal ion solutions and complex metal ion solutions were constructed to obtain standard curves for metal ions.

[0009] S4. The test solution is identified by linear discriminant analysis and hierarchical cluster analysis to obtain the types of metal ions in the test solution. The concentration of metal ions in the test solution is calculated by the standard curve of the corresponding metal ions.

[0010] Based on the above technical solution, the present invention can be further improved as follows:

[0011] Furthermore, the peptides are small molecule peptides containing thiol groups, the amides are formamides, and the metal salts are water-soluble manganese salts or water-soluble zinc salts.

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

[0013] Each metal ion possesses unique inherent properties such as electronic structure, electronegativity, ionic radius, and coordination ability. These properties determine the interaction mode and strength between metal ions and carbon dots, thus affecting the overall performance of doped carbon dots. Different metal ions can alter the electronic and band structures of carbon dots, resulting in varying effects on their fluorescence and consequently influencing their optical, electrical, and catalytic properties. Metal ions can also act as active centers or catalytic sites, promoting the activity and selectivity of carbon dots in specific reactions. Based on this, metal-doped carbon dots were synthesized by selecting manganese ions with moderate coordination ability and zinc ions with strong coordination ability as doping metals. The doping of zinc and manganese ions resulted in varying degrees of occupation of coordination sites on the carbon dot surface, causing subsequent addition of metal ions to compete for coordination, thereby affecting the changes in fluorescence induced by the metal ions.

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

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

[0016] Further, the drying temperature is -90--70℃, and the drying time is 70-74 h.

[0017] Further, the metal ions are Ag + , Cu 2+ , Fe 3+ , Hg 2+ , Yb 3+ , Zn 2+ , Pb 2+ , Cr 3+ , Zr 4+ , and Cd 2+ .

[0018] Further, the calculation formula of the relative fluorescence intensity is as follows:

[0019] .

[0020] The present application has the following beneficial effects: the target product is obtained by one-step solvothermal method, the synthesis is simple and easy to operate, the cost is low, the three kinds of carbon dots (CDs, Mn-CDs and Zn-CDs) obtained have unique fluorescence properties, and have different fluorescence responses to 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 application has high sensitivity and high selectivity, and under the doping of zinc and manganese ions, the three kinds of carbon dots show different fluorescence responses to different metal ions, and can distinguish and identify up to 10 kinds of metal ions (Ag + , Cu 2+ , Fe 3+ , Hg 2+ , Yb 3+ , Zn 2+ , Pb 2+ , Cr 3 + , Zr 4+ , and Cd 2+ ), and also to 5 kinds of metal ions (Zn 2+ , Ag+ , Hg 2+ , Cr 3+ and Cd 2+ ) enables quantitative detection with the ability to accurately identify and quantify metal ions in real samples. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

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

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

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

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

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

[0029] Figure 9 is the LDA standard score plot when the final concentration of metal ions is 20 μΜ;

[0030] Figure 10 is the LDA standard score plot when the final concentration of metal ions is 10 μΜ;

[0031] Figure 11 is the LDA standard score plot when the final concentration of metal ions is 5 μΜ;

[0032] Figure 12 is the LDA standard score plot of the sensor array for Pb 2+ and Ag + binary mixtures;

[0033] Figure 13 is the HCA analysis of the sensor array for Pb 2+ and Ag + binary mixtures;

[0034] Figure 14 is Zn2+ Linear fitting curve between Factor 1 and Factor 2;

[0035] Figure 15 Ag + Linear fitting curve between Factor 1 and Factor 2;

[0036] Figure 16 Cd 2+ Linear fitting curve between Factor 1 and Factor 2;

[0037] Figure 17 Hg 2+ Linear fitting curve between Factor 1 and Factor 2;

[0038] Figure 18 Cr 3+ Linear fitting curve between Factor 1 and Factor 2;

[0039] Figure 19 The detection result of the sample to be detected. DETAILED DESCRIPTION

[0040] The specific embodiments of the present application are described below to facilitate the understanding of the present application for those skilled in the art. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are conventional products that can be purchased on the market. However, it should be clear that the present application is not limited to the scope of the specific embodiments. For those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application as defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0041] Example 1

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

[0043] A preparation method of a manganese-doped carbon dot (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℃ for 7 h to obtain a yellow-green reaction solution; transferring the reaction solution into a dialysis bag with a molecular weight of 3500 KD, dialyzing for 7 days, then filtering with a filter membrane with a pore size of 0.3 μM, and vacuum drying the filtrate at -90℃ for 70 h to obtain the manganese-doped carbon dot.

[0044] A preparation method of zinc-doped carbon dots (Zn-CDs), comprising the following steps: mixing 100 mg glutathione, 10 mL formamide and 1 mg zinc chloride, and then reacting at 150 DEG C for 9 h to obtain a turquoise reaction solution; transferring the reaction solution into a dialysis bag with a 3500 KD to dialyze for 6 days, then filtering with a filter membrane with a pore size of 0.2 mu M, and placing the filtrate in a vacuum dryer at-70 DEG C for 74 h to obtain the zinc-doped carbon dots.

[0045] A metal ion detection method based on a three-fluorescent carbon dot sensing array, comprising the following steps:

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

[0047] S2, the linear discriminant analysis and hierarchical cluster analysis are used to discriminate the to-be-detected solution to obtain the kind of metal ion in the to-be-detected solution, and the concentration of the metal ion in the to-be-detected solution is calculated through the standard curve of the corresponding metal ion.

[0048] Example 2

[0049] A preparation method of bare carbon dots (CDs), a synthesis flow chart is shown as Figure 1 , comprising the following steps: mixing 0.7 g glutathione and 70 mL formamide, and then reacting at 160 DEG C for 8 h to obtain a dark green reaction solution; transferring the reaction solution into a dialysis bag with a 3500 KD to dialyze for 6 days, replacing water 4 times a day, then filtering with a filter membrane with a pore size of 0.22 mu M, and placing the filtrate in a vacuum dryer at-80 DEG C for 72 h to obtain the bare carbon dots (CDs).

[0050] A manganese-doped carbon dot (Mn-CDs), the difference between its preparation method and the preparation method of the bare carbon dots (CDs) of the embodiment is that the 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 that of the bare carbon dots, and the manganese-doped carbon dots (Mn-CDs) are obtained.

[0051] A zinc-doped carbon dot (Zn-CDs) is prepared by a method different from that of the bare carbon dot (CDs) in the embodiment, which is that zinc chloride is added as a raw material, i.e., 0.7 g of glutathione, 70 mL of formamide and 10 mg of zinc chloride are mixed, and the rest of the preparation process is the same as that of the bare carbon dot.

[0052] The optical properties of the bare carbon dot (CDs), manganese-doped carbon dot (Mn-CDs) and zinc-doped carbon dot (Zn-CDs) are explored. The results are shown in Figures 3-5 , and the inserted figure is a photo of the carbon dot solution under visible light (left) and 405 nm ultraviolet light (right). The CDs, Mn-CDs and Zn-CDs solutions show red, light red and bright red fluorescence under ultraviolet light, respectively. In the fluorescence spectrum, the maximum excitation wavelength of CDs is 420 nm, and there are emissions at 658 nm and 687 nm; the optimal excitation wavelength of Mn-CDs is 420 nm, and there are emission peaks at 658 nm and 687 nm; Zn-CDs have emission and excitation characteristic peaks at 658 nm and 420 nm. The three kinds of carbon dots have absorption at 420 nm and 640 nm, but the absorption peak intensities are quite different.

[0053] A metal ion detection method based on a three-fluorescent carbon dot sensing array, as shown in Figure 2 , comprises the following steps:

[0054] S1, the bare carbon dot, manganese-doped carbon dot and zinc-doped carbon dot solutions are respectively subjected to fluorescence detection to obtain the original fluorescence intensity F0, then the bare carbon dot, manganese-doped carbon dot and zinc-doped carbon dot solutions are respectively mixed with different kinds of metal ion (Ag + , Cu 2+ , Fe 3+ , Hg 2+ , Yb 3+ , Zn 2+ , Pb 2+ , Cr 3+ , Zr 4+ and Cd 2+ ) standard solutions, the final concentration of the metal ion is 10 μM, the final concentration of the CDs is 10 μg / mL, the fluorescence intensity F of different kinds of metal ions is obtained; finally, the relative fluorescence intensity is obtained by normalization, the relative fluorescence intensity is converted into a typical discriminant factor by using linear discriminant analysis, a fluorescence sensing array and a database of single metal ion solution and composite metal ion solution are constructed, and a standard curve of the metal ion is obtained; the calculation formula of the relative fluorescence intensity is: ;

[0055] 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 min, then the solution is detected by using a fluorescence spectrometer and the fluorescence spectrum is recorded, the excitation light is 420 nm, the voltage is 400 V, and the excitation and emission slits are both 5.0 nm. 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 min, then the fluorescence intensity F of the solution at 687 nm is recorded by using an enzyme-labeled instrument, and each group is repeated 5 times.

[0056] S2, discriminating the to-be-tested solution by linear discriminant analysis and hierarchical cluster analysis to obtain the type of metal ion in the to-be-tested solution, and calculating the concentration of the metal ion in the to-be-tested solution by the standard curve of the corresponding metal ion.

[0057] By Figures 6-8 It can be observed that the fluorescence spectra of the three types of carbon dots all change to different degrees, for example, Hg 2+ can greatly quench the fluorescence of the three types of CDs, while Ag + can quench the fluorescence of the three types of CDs to a certain extent, and Cu 2+ has a slightly weaker quenching ability on Zn 2+ , but can effectively quench the fluorescence of CDs and Mn-CDs, and Zn 2+ can greatly enhance the emission peak intensity of CDs and Mn-CDs at 658 nm, but has little effect on Zn-CDs; it proves the feasibility of using the three types of carbon dots as sensing probes for heavy metal ion identification.

[0058] 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 types of fluorescent carbon dots prepared in Example 2.

[0059] Experimental Example 1 Identification and quantitative ability of array sensing

[0060] 1. Qualitative identification

[0061] In order to verify the identification ability of the array sensing constructed by the application, 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+100 μL each of three fluorescent probes (CDs, Mn-CDs, and Zn-CDs, all at a concentration of 10 μg / mL) were mixed with 100 μL each of the three final concentrations of 5, 10, and 20 μM 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, resulting in a training data matrix of 3 carbon dots × 10 metal ions × 5 replicates. Figures 9-11 As shown, when the final concentration of metal ions is 5, 10 and 20 μM, the array sensor has good recognition ability. All 10 heavy metal ions detected were correctly identified, with a recognition accuracy of 100%.

[0062] To ensure the ability to identify mixed metal ions, array sensing was used to measure mixed heavy metal ions. The specific steps were as follows: Ag with different molar ratios... + and Pb 2+ Binary mixture (Ag) + Pb 2+ 100 μL each of the ratios 9:1, 7:3, 5:5, 3:7, and 1:9 (total metal ion concentration of 10 μM) were mixed with 100 μL each of three fluorescent probes (CDs, Mn-CDs, and Zn-CDs, all at a concentration of 10 μg / mL). The fluorescence response was recorded five times, and the mixtures were then analyzed. The results are as follows: Figure 12 As shown, pure Ag can be identified in the LDA plot. + and Pb 2+ And its mixture, and precisely arranged in order of molar ratio on Factor1. For example... Figure 13 As shown, the HCA results also showed that each of the 35 samples was accurately classified into the corresponding category, indicating that the array sensor has a good ability to distinguish between different concentrations of mixed metal ions. The CDs-based sensor array can identify mixtures containing mixed metal ions.

[0063] 2. Quantitative detection

[0064] To determine the method of the present invention for detecting metal ions (Zn) 2+ Ag + Hg 2+ Cr 3+ and Cd 2+ The sensitivity of Zn was assessed by correlating the value of factor 1 in a typical scoring plot with a series of Zn concentrations. 2+ (0.25-2.5μM), Ag + (1-2.5μM), Hg 2+ (0.05-1000μM), Cr 3+ (0.01-0.25 μM) and Cd 2+(0.05-2.5 μM), and a good linear relationship between Factor 1 and the concentration of metal ions was verified, as shown in Figures 14-18 Fig. 2, indicating that the fluorescence sensing array can also be used for quantitative detection of specific heavy metal ions. Subsequently, a database containing 5 metal ions was constructed to quantitatively analyze heavy metal ions, each curve started at 0 μM and connected to the data points representing specific heavy metal ions at corresponding concentrations, showing the continuous spatial changes of individual heavy metal ions with increasing concentrations. The results of quantitative detection of metal ions by Factor 1 scores are shown in Table 1.

[0065] Table 1. Results of quantitative detection of metal ions by Factor 1 scores

[0066]

[0067] Result analysis: As can be seen from Table 1, the content of specific heavy metal ions can be qualitatively identified and quantitatively detected by the method of the present application, and the detection limit can be as low as 11.46 nM, which can realize accurate identification and detection of heavy metal ions.

[0068] Experimental Example 3. Detection of actual samples

[0069] 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, and two different concentrations of metal ions were prepared as 4 samples, labeled as samples 3-6. The fluorescence signals of samples 1-6 were then detected by ICP and analyzed by LDA. The ICP results are shown in Tables 2 and 3, and the LDA analysis is shown in Figure 19 Fig. 3, sample 1, 2 was classified as Zn 2+ , sample 3, 4 was classified as Cd 2+ , and sample 5, 6 was classified as Hg 2+ . Finally, the samples 1-6 were quantitatively detected according to the linear equation of Figures 14-18 , which was roughly consistent with the ICP results, with a relative deviation of 3.69-16.82%, indicating that the present application has the ability to accurately identify and quantify metal ions in actual samples.

[0070] Table 2. Detection results of actual samples

[0071]

[0072] Table 3. Detection results of spiked samples

[0073]

Claims

1.A method for detecting metal ions based on three fluorescent carbon dots sensing array, characterized in that, The method comprises the following steps: S1, mixing the peptide substance and the amide substance, and then reacting at 150-170 DEG C for 7-9 hours to obtain a reaction solution; the reaction solution is dialyzed for 6-7 days, then filtered, and the filtrate is freeze-dried to obtain naked carbon dots; the ratio of the peptide substance to the amide substance is 100-200 mg: 10-20 mL; the peptide substance is a small molecule peptide containing sulfydryl, and the amide substance is formamide; S2, mixing the peptide substance, the amide substance, the water-soluble manganese salt and the water-soluble zinc salt, and then reacting at 150-170 DEG C for 7-9 hours to obtain a reaction solution; the reaction solution is dialyzed for 6-7 days, then filtered, and the filtrate is freeze-dried to obtain manganese-doped carbon dots and zinc-doped carbon dots; the ratio of the peptide substance, the amide substance, the water-soluble manganese salt and the water-soluble zinc salt is 100-200 mg: 10-20 mL: 1-3 mg; S3, the manganese-doped carbon dot solution, the zinc-doped carbon dot solution and the bare carbon dot solution are respectively subjected to fluorescence detection to obtain original fluorescence intensity F0, then the manganese-doped carbon dot solution, the zinc-doped carbon dot solution and the bare carbon dot solution are respectively mixed with different kinds of metal ion standard solutions to obtain fluorescence intensity F of different kinds of metal ions; finally, relative fluorescence intensity is obtained through normalization , the relative fluorescence intensity is converted into a typical discriminant factor by using linear discriminant analysis, a fluorescence sensing array and a database of single metal ion solution and composite metal ion solution are constructed, and a standard curve of metal ions is obtained; S4, the type of metal ions in the test solution is obtained by linear discriminant analysis and hierarchical cluster analysis, and the concentration of metal ions in the test solution is calculated by the standard curve of the corresponding metal ions. 2.The method of claim 1, wherein the three kinds of fluorescent carbon dots are C-dots, C-dots and C-dots. The peptide substance is glutathione, the water-soluble manganese salt is manganese perchlorate hexahydrate, and the water-soluble zinc salt is zinc chloride. 3.The method of claim 1, wherein the three kinds of fluorescent carbon dots are C-dots, C-dots and C-dots. The pore size of the filter membrane used for filtering is 0.2-0.3 mu m. 4.The method of claim 1, wherein the three kinds of fluorescent carbon dots are C-dots, C-dots and C-dots. The drying temperature is-90 to-70 DEG C, and the drying time is 70-74 hours. 5.The method of claim 1, wherein the three kinds of fluorescent carbon dots are C-dots, C-dots and C-dots. the metal ion is Ag + , Cu 2+ , Fe 3+ , Hg 2+ , Yb 3+ , Zn 2+ , Pb 2+ , Cr 3+ , Zr 4+ , and Cd 2+ . 6.The method of claim 1, wherein the three kinds of fluorescent carbon dots are C-dots, C-dots and C-dots. The relative fluorescence intensity The calculation formula is: 。

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