CN115386376B - Nano platinum composite material based on carbon quantum dot assembly and preparation method and application thereof - Google Patents
Nano platinum composite material based on carbon quantum dot assembly and preparation method and application thereof Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention discloses a nano platinum composite material based on a carbon quantum dot assembly and a preparation method and application thereof. The method takes Polyethyleneimine (PEI), dopamine (DA) and carbon quantum dots containing aldehyde (CHO-CQDs) as raw materials, and forms an assembly (N/O-s-CQDs) through chemical reaction 3+ The effect can generate obvious fluorescence quenching effect, and the method has high sensitivity and can quickly detect the iron ions Fe in the sample 3+ Concentration and the like. The nano platinum composite material (Pt @ N/O-s-CQDs) based on the carbon quantum dot assembly is prepared by reducing platinum ions in situ by using active groups in the N/O-s-CQDs. The composite material is illuminated by near infrared light with the wavelength of 808 nm, has the photo-thermal effect, combines the anti-oxidation characteristic of nano platinum therein, and can play an application value in the antibacterial field.
Description
Technical Field
The invention relates to the technical field of nano composite materials, in particular to a nano platinum composite material based on a carbon quantum dot assembly and a preparation method and application thereof.
Background
Fe 3+ The ions play a crucial role in various biological activities of the human body, such as oxygen transport, enzyme catalysis, DNA synthesis, electron transport, formation of free radicals in enzyme-based reactions, cell metabolism, etc., and are deficient in Fe 3+ Ions can cause many diseases such as heart failure, anemia, tissue damage, parkinson's disease, kidney and liver damage, alzheimer's disease, cancer, arthritis, and diabetes. Fe 3+ There are many conventional measurement methods such as inductively coupled plasma spectrophotometry (ICPMS), colorimetry, x-ray fluorescence spectrophotometry, electrochemistry, atomic absorption/emission spectrometry, etc., however, these methods are time consuming, complicated to operate, and require expensive instruments and complicated sample pretreatment procedures.
The chemical sensor based on the nano material has excellent electronic, magnetic and optical properties, and can be used for detecting the amount of heavy metal in aqueous solution, namely Fe 3+ Have developed a variety of nanoparticle-based fluorescent probes such as metal organic frameworks, gold nanoclusters, and the like. At present, the research on nano fluorescent probes is increasing, and the Fluorescence (FL) established based on these probes draws great attention from researchers due to its advantages of excellent sensitivity, detection capability at a minute concentration, low cost, short response time, and the like. In recent years, carbon Quantum Dots (CQDs) as a new fluorescent nanoparticle have a generally spherical structure, a particle size of less than 10 nm, strong and stable fluorescence, and compared with organic dyes, quantum dots and noble metal nanoparticles, CQDs have excellent water solubility, good light stability, super-strong biocompatibility, low toxicity and the like, and have complex surfaces, generally have hydroxyl groups, carboxyl groups or amino groups, and can coordinate with metal ions to cause fluorescence change, but are often influenced by interferents in the detection process because of no specifically identified groups.
In view of the above, there is a need to develop a new carbon quantum dot composite material and ferric ion Fe thereof 3+ The application in the aspect of rapid detection.
Disclosure of Invention
The invention aims to provide a preparation method and application of a novel carbon quantum dot assembly aiming at the defects of the prior art, and the novel carbon quantum dot assembly has the advantages of rapid detection of ferric ions and high accuracy.
The technical scheme adopted by the invention to achieve the aim is as follows:
the invention firstly prepares nano platinum composite material Pt @ N/O-s-CQDs, and concretely comprises the following steps:
1) Dissolving 30-60 mg of carbon quantum dot assembly N/O-s-CQDs into 30 mL of pure water, and fully dissolving by ultrasonic to obtain a solution C;
2) And (3) dropwise adding 20 mL of 60-120 nM chloroplatinic acid solution into the solution C, reacting at 25-80 ℃ for 4-8 h, dialyzing the obtained solution in pure water for a certain time, performing suction filtration, freeze drying to obtain a nano platinum composite material Pt @ N/O-s-CQDs, and storing for later use.
Preferably, the dialysis treatment in step 2) is performed in a dialysis bag of 1000/2000 Da for 12-48 h.
Wherein the preparation process of the carbon quantum dot assembly N/O-s-CQDs in the step 1) comprises the following steps:
firstly, 0.12-0.48 g of CHO-CQDs is dissolved in 40 mL of pure water and fully dissolved by ultrasonic to obtain a solution A; dissolving 2.24-3.20 g of Polyethyleneimine (PEI) and 0.1-0.0062 g of Dopamine (DA) into 40 mL of ethanol, and performing ultrasonic full dissolution to obtain a solution B; and then dropwise adding the solution B into the solution A, reacting for 6-10 h to obtain a mixed solution, dialyzing in pure water for a certain time, and then carrying out suction filtration and freeze drying to obtain the carbon quantum dot assembly N/O-s-CQDs.
Preferably, the dialysis treatment in the preparation process of the carbon quantum dot assemblies N/O-s-CQDs is carried out in a dialysis bag of 1000/2000/8000-14000 Da for 12-48 h.
The invention applies the prepared novel carbon quantum dot assembly N/O-s-CQDs to the detection of ferric ions, and specifically comprises the following steps: dissolving the prepared novel carbon quantum dot assembly N/O-s-CQDs in ultrapure water to prepare solution D, and testing the fluorescence intensity of the solution D and marking as F 0 (ii) a Then the different concentrations areDegree of Fe 3+ Respectively mixing the solution D with the solution D to respectively obtain corresponding mixed solutions, and performing fluorescence test to obtain fluorescence intensity values of the mixed solutions, and marking the fluorescence intensity values as F; with Fe 3+ Concentration is abscissa, fluorescence quenching rate (1-F/F) 0 ) Performing linear fitting to obtain a regression equation y1= k1x1+ b1 as an ordinate, wherein y1 is a fluorescence quenching rate, and x1 is Fe 3+ The value of k1 is the slope, and the value of b1 is the intercept; will contain Fe 3+ The solution is mixed with the solution D to obtain a Fe-N/O-s-CQDs mixed solution for fluorescence test to obtain a fluorescence intensity value of the Fe-N/O-s-CQDs mixed solution, the fluorescence intensity value is substituted into a linear regression equation y1= k1x1+ b1, and Fe is obtained by calculation 3+ The concentration of (c).
Preferably, the mass concentration of the solution D is 10-20 mg/mL.
Preferably, said different concentrations of Fe 3+ Fe in solution 3+ The concentration is 10-400. Mu.M.
Compared with the prior art, the invention has the following technical advantages:
1. the invention takes Polyethyleneimine (PEI), dopamine (DA) and carbon quantum dots containing aldehyde groups (CHO-CQDs) as raw materials, forms a novel carbon quantum dot assembly (N/O-s-CQDs) through chemical reaction, introduces high-activity functional groups (such as amino) simply and greenly, takes the carbon quantum dot assembly (N/O-s-CQDs) as a closed fluorescent probe, and realizes Fe on the basis of fluorescence quenching effect 3+ The method has the advantages of high sensitivity, good selectivity, strong anti-interference, low cost and the like, and the synthetic scheme is green and environment-friendly and has good biocompatibility.
2. By utilizing the residual active groups in the assembly (N/O-s-CQDs), platinum ions are reduced in situ to generate nano platinum (Pt NPs) under a certain temperature condition and are relatively uniformly loaded in the assembly, and a covalent modification method is adopted to carry out chemical reaction by adding functional molecules on the groups on the surfaces of the CQDs, so that the nano platinum composite material (Pt @ N/O-s-CQDs) based on the carbon quantum dot assembly is prepared. The composite material has a potential photo-thermal effect, when the composite material is prepared into an aqueous solution with a certain concentration, the temperature can be rapidly raised within a certain time, and the composite material has a potential application value in the antibacterial field by combining the antioxidation characteristic of nano platinum, so that the synthesis scheme is green and environment-friendly, and the biocompatibility is good.
The above is an overview of the technical solutions of the present invention, and the present invention is further described below with reference to the accompanying drawings and the detailed description thereof.
Drawings
FIG. 1 is an FTIR infrared detection of N/O-s-CQDs and Pt @ N/O-s-CQDs of the present example;
FIG. 2 is a TEM image of Pt @ N/O-s-CQDs of the present example;
FIG. 3 is an HRTEM image of Pt @ N/O-s-CQDs of the present example;
FIG. 4 shows the fluorescence intensity change of N/O-s-CQDs of this example with Fe 3+ The concentration is in the range of 10-400 μ M;
FIG. 5 is a diagram of detection of Fe by N/O-s-CQDs 3+ A schematic diagram of (a);
FIG. 6 is a diagram of detection of Fe by N/O-s-CQDs 3+ Interference immunity schematic diagram of (a);
FIG. 7 is a graph showing fluorescence intensity measured by N/O-s-CQDs.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the following embodiments are described in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The method comprises three aspects, namely synthesizing N/O-s-CQDs, and forming assemblies (N/O-s-CQDs) by taking Polyethyleneimine (PEI), dopamine (DA) and carbon quantum dots (CHO-CQDs) containing aldehyde groups as raw materials through chemical reaction. Secondly, preparing aqueous solution with proper concentration by using N/O-s-CQDs, adding iron ion solution with certain concentration to prepare mixed solution, and detecting Fe by using a fluorescence spectrophotometry 3+ The concentration of (c).
Thirdly, synthesizing a nano platinum composite material Pt @ N/O-s-CQDs, utilizing residual active groups in the assembly (N/O-s-CQDs), carrying out in-situ reduction on platinum ions to generate nano platinum (Pt NPs) under a certain temperature condition, and relatively uniformly loading the nano platinum (Pt NPs) in the assembly to prepare the nano platinum composite material (Pt @ N/O-s-CQDs) based on the carbon quantum dot assembly;
the synthesis route of the assembly (N/O-s-CQDs) is that CHO-CQDs (0.12-0.48 g), polyethyleneimine (PEI) (2.24-3.20 g) and Dopamine (DA) (0.1-0.0062 g) are used as raw materials, reaction is carried out for a certain time (6-10 h) at normal temperature, and the obtained crude product is purified by a dialysis bag of 1000/2000/8000-14000 Da and then freeze-dried for later use. The specific implementation process is as follows:
example 1
The preparation method of the assembly N/O-s-CQDs of the embodiment comprises the following steps:
firstly, 0.32 g of CHO-CQDs is dissolved in 40 mL of pure water, and the solution A is obtained by ultrasonic full dissolution; dissolving 2.50 g of Polyethyleneimine (PEI) and 0.025 g of Dopamine (DA) in 40 mL of ethanol, and sufficiently dissolving by ultrasonic to obtain a solution B; and then dropwise adding the solution B into the solution A, reacting for 6 hours to obtain a mixed solution, transferring the mixed solution into a 2000 Da dialysis bag, dialyzing for 48 hours, carrying out suction filtration by using a 0.22 mu m organic system filter membrane, carrying out rotary evaporation at 60 ℃, freezing for 4 hours at-60 ℃, carrying out vacuum drying for 24 hours to obtain a dark brown powdery assembly (N/O-s-CQDs), and storing for later use at 2-8 ℃. Because a certain amount of DA is added into the assembly reaction system, the DA participates in the assembly reaction and can also perform self-polymerization to form an indole ring structure (shown in figure 1), and the fluorescence intensity of the correspondingly prepared N/O-s-CQDs aqueous solution is obviously stronger than that of N-s-CQDs with the same concentration. (see FIG. 7)
Example 2
The preparation of the assemblies N/O-s-CQDs of this example was carried out by changing the amount of dopamine to 0.05 g as compared with example 1.
Example 3
The preparation of the assemblies N/O-s-CQDs of this example was carried out by changing the amount of CHO-CQDs to 0.48 g as compared with example 1.
Example 4
Compared with the preparation method of the assembly N/O-s-CQDs in the example 1, the reaction time is changed into 10 hours under the normal temperature condition.
Example 5
The preparation of the assemblies N/O-s-CQDs of this example was carried out by changing the dialysis time to dialysis 24 hours, as compared with example 1.
Example 6
As an application example, the method of detecting Fe by fluorescence spectrophotometer 3+ In this embodiment, the application of the prepared novel carbon quantum dots N/O-s-CQDs to the detection of ferric ions specifically includes the following steps: the novel carbon quantum dots N/O-s-CQDs prepared in example 1 were dissolved in ultrapure water to prepare a solution D having a concentration of 10 mg/mL, and 500. Mu.M of Fe 3+ Mixing the solution with the solution D to obtain a mixed solution E1, performing fluorescence test on the mixed solution E1, and recording the fluorescence intensity of the mixed solution as F 0 (ii) a Then respectively mixing 0-550 mu M Fe3+ solution with the mixed solution E1 to obtain corresponding mixed solution E, and carrying out fluorescence test to obtain the fluorescence intensity value of each mixed solution, and marking as F; with Fe 3+ Concentration as abscissa, fluorescence quenching rate (1-F/F) 0 ) Performing linear fitting to obtain a regression equation y1= k1x1+ b1 as an ordinate, wherein y1 is a fluorescence quenching rate, and x1 is Fe 3+ The value of k1 is the slope, and the value of b1 is the intercept; will contain Fe 3+ The solution is mixed with the solution E1 to obtain a Fe-N/O-s-CQDs mixed solution for fluorescence test, the fluorescence intensity value of the Fe-N/O-s-CQDs mixed solution is obtained, the fluorescence intensity value is substituted into a linear regression equation y1= k1x1+ b1, and Fe is obtained through calculation 3+ The concentration of (c).
The synthesis of nano platinum composite material Pt @ N/O-s-CQDs adopts a synthetic route that an assembly N/O-s-CQDs (30-60 mg) and chloroplatinic acid (60-120 nM) are used as raw materials, the raw materials react for a certain time (4-8 h) at a certain temperature (25-80 ℃), and the obtained crude product is purified by a dialysis bag of 1000/2000 and then is freeze-dried for standby. The specific implementation process is as follows:
example 7
The preparation method of the nano platinum composite material Pt @ N/O-s-CQDs of the embodiment comprises the following steps:
1) Dissolving 30 mg of carbon quantum dots N/O-s-CQDs into 30 mL of pure water, and fully dissolving by ultrasonic to obtain a solution C;
2) Dropwise adding 20 mL of 105 nM chloroplatinic acid solution into the solution C, continuously reacting for 8 h at 80 ℃, transferring the obtained solution into a dialysis bag of 2000 Da, dialyzing for 48 h in pure water, then carrying out rotary evaporation at 80 ℃ for a certain time, freezing for 4 h at-60 ℃, and carrying out vacuum drying for 24 h to obtain the nano platinum composite material (Pt @ N/O-s-CQDs), and storing for later use at 2-8 ℃. Wherein the nano platinum is spherical, and has an average particle diameter of about several nanometers (see attached figures 2 and 3).
Example 8
Compared with the preparation method of the nano platinum composite material Pt @ N/O-s-CQDs in the embodiment 7, the concentration of the chloroplatinic acid is changed, and the solution is specifically a solution of 20 mL and 90 nM chloroplatinic acid.
Example 9
Compared with the preparation method of the nano platinum composite material Pt @ N/O-s-CQDs in the embodiment 7, the concentration of the chloroplatinic acid is changed, and the solution is specifically 20 mL and 120 nM chloroplatinic acid.
Example 10
Compared with the preparation method of the example 7, the preparation method of the nano platinum composite material Pt @ N/O-s-CQDs changes the dosage of the N/O-s-CQDs, and specifically 60 mg of the N/O-s-CQDs is dissolved in 30 mL of water.
Example 11
Compared with the preparation method of the nano platinum composite material Pt @ N/O-s-CQDs in the embodiment 7, the reaction temperature is changed, and specifically, the reaction is continued for 6 hours at 60 ℃.
Example 12
Compared with the preparation method of the example 7, the preparation method of the nano platinum composite material Pt @ N/O-s-CQDs changes the reaction time, and particularly continuously reacts for 6 hours at 80 ℃.
Example 13
In this embodiment, the nano platinum composite material Pt/N-s-CQDs based on the carbon quantum dot assembly prepared by the method is applied to photo-thermal, and specifically includes the following steps: the nano platinum composite material Pt/N-s-CQDs based on the carbon quantum dot assembly prepared in the example 7 is dissolved in ultrapure water to prepare a solution D with the concentration of 1 mg/mL. The temperature of the sample is gradually increased to be stable in a certain time by adopting a photo-thermal instrument with the wavelength of 808 nm, and the temperature change of the sample is tested.
The products of the examples of the present invention were tested as shown in FIG. 1 of the specification, and N/O-s-CQDs was 3380 cm -1 The peak at (A) is ascribed to the stretching vibration of O-H bond and N-H bond in amide bond, 1654 cm -1 The peak at (A) was ascribed to the stretching vibration of C = O bond in amide bond, 1572 cm -1 The peak at (A) is attributed to the characteristic peak of the dihydroxyindole moiety in polydopamine, 1479 cm -1 The peak belongs to the stretching vibration of the C-N bond, which can show that the CHO-CQDs react with PEI and DA to introduce-CONH-into the assembly N/O-s-CQDs; comparing FTIR graphs of N/O-s-CQDs with Pt @ N/O-s-CQDs, the characteristic peaks are slightly shifted because of the interaction of amine groups, carbonyl groups and the like in the assembly and the loaded nano platinum.
As shown in the attached drawings of the specification, FIGS. 2 and 3, TEM and HRTEM images of Pt @ N/O-s-CQDs show that the Pt (IV) is reduced to the simple substance Pt and is uniformly loaded in the assembly, and the pattern has a 0.217 nm lattice fringe pattern corresponding to the Pt (100) lattice fringe.
As shown in FIG. 4 of the accompanying drawings, the fluorescence quenching rate is 1-F/F 0 With Fe 3+ Graph of concentration dependence of fluorescence quenching rate on Fe 3+ The concentration is in a good linear relationship in the range of 10-400. Mu.M. Linear equation of 1-F/F 0 =0.00134C Fe3+ +0.05249, good linear correlation, correlation coefficient (R) 2 ) And is 0.99933.
Also shown in the attached figure 5 of the specification, the detection of the Fe by N/O-s-CQDs 3+ In the selectivity of (1), 500 mu M of Fe is respectively added into the N/O-s-CQDs solution 3+ 、Ni 2+ 、Na + 、Mg 2+ 、K + 、Zn 2+ 、Ba 2+ 、Hg 2+ 、Mn 2+ 、 Ca 2+ 、Pb 2+ Change of fluorescence intensity before and after the metal ions are plasma-generated. Degree of quenching of fluorescence (in F/F) when other common metal ions are added to N/O-s-CQDs solution 0 Expressed) has almost no influence, but only Fe is added thereto 3+ Then, the fluorescence of the N/O-s-CQDs solution is remarkably reduced, and fluorescence quenching is generated.
Also as illustratedAs shown in figure 6 of the attached drawings, the detection of Fe by N/O-s-CQDs 3+ The anti-interference performance of the system. The data of group 1 is a control group, from the data of group 2 (i.e. from the 3 rd histogram), the left side of each data group shows the experimental results corresponding to the N/O-s-CQDs solution added with a common metal ion, and the right side shows that a common metal ion and Fe are added simultaneously 3+ The results of the experiments corresponding to the mixed N/O-s-CQDs solution. The results show that except for Fe 3+ The other 10 common metal ions do not have a particularly large influence on the fluorescence intensity of N/O-s-CQDs. However, when Fe 3+ When the fluorescent dye is added into N/O-s-CQDs solution, the fluorescence intensity is obviously reduced, and fluorescence quenching is generated. It can be concluded that N/O-s-CQDs are responsible for Fe 3+ Has good selectivity.
As shown in the attached figure 7 of the specification, the assembly N/O-s-CQDs is prepared according to different mass ratios of PEI and DA, when mPEI: mDA = 100: 1, the fluorescence intensity of the obtained N/O-s-CQDs is the highest, and the time of the same concentration is several times that of the assembly N-s-CQDs without DA participation.
Variations and modifications to the above-described embodiments may occur to those skilled in the art, which fall within the scope and spirit of the above description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and modifications and variations of the present invention are also intended to fall within the scope of the appended claims.
Claims (8)
1. A preparation method of a nano platinum composite material Pt @ N/O-s-CQDs based on a carbon quantum dot assembly is characterized by comprising the following steps:
1) Dissolving 30-60 mg of carbon quantum dot assembly N/O-s-CQDs into 30 mL of pure water, and fully dissolving by ultrasonic to obtain a solution C;
2) Dropwise adding 20 mL of 60-120 nM chloroplatinic acid solution into the solution C, reacting at 25-80 ℃ for 4-8 h, dialyzing the obtained solution in pure water for a certain time, filtering, and freeze-drying to obtain a nano platinum composite material Pt @ N/O-s-CQDs, and storing for later use;
the preparation process of the carbon quantum dot assembly N/O-s-CQDs in the step 1) comprises the following steps:
firstly, 0.12-0.48 g of aldehyde-containing carbon quantum dots CHO-CQDs are dissolved in 40 mL of pure water, and the solution A is obtained by ultrasonic full dissolution; dissolving 2.24-3.20 g of polyethyleneimine PEI and 0.1-0.0062 g of dopamine DA in 40 mL of ethanol, and sufficiently dissolving by ultrasonic to obtain a solution B; and then dropwise adding the solution B into the solution A, reacting for 6-10 h to obtain a mixed solution, dialyzing in pure water for a certain time, and then performing suction filtration and freeze drying to obtain the carbon quantum dot assembly N/O-s-CQDs.
2. The method for preparing nano platinum composite material Pt @ N/O-s-CQDs based on carbon quantum dot assembly according to claim 1, wherein the dialysis treatment in the step 2) is performed in a dialysis bag of 2000 Da for 12-48 h.
3. The method for preparing nano platinum composite material Pt @ N/O-s-CQDs based on carbon quantum dot assembly as claimed in claim 1, wherein the dialysis treatment in the preparation process of the carbon quantum dot assembly N/O-s-CQDs is carried out in a dialysis bag of 2000 Da for 12-48 h.
4. A nano platinum composite material Pt @ N/O-s-CQDs based on a carbon quantum dot assembly is characterized by being prepared by the preparation method of any one of claims 1-3.
5. The application of the carbon quantum dot assembly N/O-s-CQDs is characterized in that the carbon quantum dot assembly N/O-s-CQDs in the claim 1 or 3 are applied to the detection of ferric ions.
6. The use of the carbon quantum dot assemblies N/O-s-CQDs according to claim 5, wherein the use for the detection of ferric ions comprises the steps of:
s1, dissolving the prepared carbon quantum dot assembly N/O-S-CQDs in ultrapure water to prepare a solution D, and testing the fluorescence intensity of the solution D, wherein the fluorescence intensity is marked as F 0 ；
S2, then adding Fe with different concentrations 3+ Respectively mixing the solution with the solution D to respectively obtain corresponding mixed solutions, and performing fluorescence test to obtain fluorescence intensity values of the mixed solutions, and marking the fluorescence intensity values as F;
s3, with Fe 3+ Concentration is abscissa, fluorescence quenching rate (1-F/F) 0 ) Performing linear fitting to obtain a regression equation y1= k1x1+ b1 as an ordinate, wherein y1 is a fluorescence quenching rate, and x1 is Fe 3+ The value of k1 is the slope, and the value of b1 is the intercept;
s4, fe is to be detected 3+ The solution is mixed with the solution D to obtain a Fe-N/O-s-CQDs mixed solution for fluorescence test to obtain a fluorescence intensity value of the Fe-N/O-s-CQDs mixed solution, the fluorescence intensity value is substituted into a linear regression equation y1= k1x1+ b1, and Fe is obtained by calculation 3+ The concentration of (c).
7. The use of carbon quantum dot assemblies N/O-s-CQDs as claimed in claim 6, wherein said different concentrations of Fe 3+ Fe in solution 3+ The concentration is 10-400. Mu.M.
8. The use of carbon quantum dot assemblies N/O-s-CQDs as claimed in claim 6, wherein the mass concentration of solution D is 10-20 mg/mL.
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