Preparation method and application of carbonized polymer nanodot for chemiluminescence detection

Abstract

The invention discloses a preparation method and application of carbonized polymer nanodots for chemiluminescence detection. The preparation method comprises the following steps: 1) dissolving a carbon source in water to form a uniform solution; 2) slowly adding a nitrogen source into the uniform solution to form a mixed solution; 3) heating the obtained mixed solution for reaction; 4) after the water is completely evaporated, changing the set temperature of the heating reaction, so that the yellow liquid is converted into a dark brown soft semi-solid; 5) cooling the dark brown soft semisolid to room temperature, and grinding into powder to obtain carbonized polymer nanodots; the step 3) and the step 4) are both carried out under ambient pressure. The carbonized polymer nanodots are prepared by adopting a more energy-saving and environment-friendly solvothermal method, the preparation process is simple, high temperature and high pressure are not needed, the particle size is smaller, the distribution is more uniform, and the shape is more regular.

Description

Technical Field

[0001] The present invention relates to the technical field of chemiluminescence detection, and in particular to a method for preparing carbonized polymer nanodots for chemiluminescence detection and an application thereof. Background Art

[0002] Chemiluminescence reaction systems are widely used in a variety of tests such as biological analysis and environmental analysis because of their advantages such as easy operation, high sensitivity and good selectivity. However, due to the poor selectivity of existing chemiluminescence systems, the extremely fast chemiluminescence rate, and the decay of the peak luminescence intensity in a short time, the method has poor reproducibility and low stability, and there are fewer products that can be generated in the excited state and fewer specific luminescent reagents. Traditional organic fluorescent dyes and semiconductor quantum dots are highly toxic, which limits the scope of application of chemiluminescence analysis.

[0003] Carbonized polymer nanodots are emerging fluorescent nanomaterials that are closely connected to strong and weak chemiluminescent systems. For example, Chen et al. (Chen H, Lin L, Lin Z, et al. Flow-injection analysis of hydrogen peroxide based on carbon nanospheres catalyzed hydrogencarbonate–hydrogen peroxide chemiluminescent reaction [J]. Analyst, 2011, 136 (9): 1957-1964.) used glucose as the carbon source and added hydrothermally synthesized carbon nanospheres to the NaHCO3-H2O2 system. They found that the chemiluminescence intensity of the system was enhanced, and established a new NaHCO3-H2O2-CNSs chemiluminescent system that can measure the H2O2 content in water samples with high sensitivity, selectivity, rapidity and repeatability, and shows good consistency with other reported chemiluminescent methods. However, the chemiluminescent signal will weaken rapidly after reaching the maximum value, which may limit the signal acquisition time and data processing. For example, Lin et al. (Lin Z, Xue W, Chen H, et al. Peroxynitrous-acid-induced chemiluminescence of fluorescent carbon dots for nitrite sensing [J]. Analytical chemistry, 2011, 83 (21): 8245-8251.) used PEC1500, glycerol and serine as precursors to synthesize carbonized polymer nanodots by microwave method, added them to NaNO2-H2O2 system, and established a new NaNO2-H2O2-CDs chemiluminescence system to measure the nitrite content in pond water, river water and milk; they proposed that electron transfer annihilation may be the main cause of chemiluminescence emission, providing new insights into the optical properties of carbonized polymer nanodots; but despite the enhanced chemiluminescence signal, the stability and duration of the chemiluminescence reaction remains a challenge.For example, Shah et al. (Shah SN, Li H, Lin JM. Enhancement of periodate-hydrogen peroxide chemiluminescence by nitrogendoped carbon dots and its application for the determination of pyrogallol and gallic acid [J]. Talanta, 2016, 153: 23-30.) found that carbonized polymer nanodots can significantly enhance the luminescence signal of the alkaline system KIO4-H2O2 system, and established a KIO4-H2O2-N-CQDs (OH-) system. The chemiluminescence mechanism of the system was studied by EPR, chemiluminescence spectroscopy and other means, and it was speculated that it was a free radical action mechanism; carbonized polymer nanodots acted as catalysts and energy acceptors in the KIO4-H2O2 system, and enhanced the chemiluminescence signal through chemical resonance energy transfer.

[0004] However, the current preparation methods of carbonized polymer nanodots have the following defects: 1) high energy consumption; 2) the reagents used may pollute the environment; 3) the gas pressure required for the reaction is not atmospheric pressure; 4) the optical properties of carbonized polymer nanodots are poor. Summary of the invention

[0005] The first technical problem to be solved by the present invention is to provide a method for preparing carbonized polymer nanodots for chemiluminescence detection. The preparation method adopts a more energy-saving and environmentally friendly solvent thermal method, and heats under ambient pressure to obtain small-sized carbonized polymer nanodots with uniform particle size, regular shape and good optical properties.

[0006] The second technical problem to be solved by the present invention is to provide an application of carbonized polymer nanodots in chemiluminescence detection.

[0007] In order to solve the above-mentioned first technical problem, the technical solution adopted by the invention is as follows:

[0008] A method for preparing carbonized polymer nanodots for chemiluminescence detection comprises the following steps:

[0009] 1) dissolving the carbon source in water to form a uniform solution;

[0010] 2) slowly adding a nitrogen source to the above uniform solution to form a mixed solution;

[0011] 3) heating the obtained mixed solution to react;

[0012] 4) After the water is completely evaporated, the set temperature of the heating reaction is changed to transform it from a yellow liquid to a dark brown soft semi-solid;

[0013] 5) cooling the dark brown soft semi-solid to room temperature and grinding it into powder to obtain carbonized polymer nanodots;

[0014] Step 3) and step 4) are both carried out under ambient pressure.

[0015] Preferably, in step 1), the carbon source is citric acid.

[0016] Preferably, in step 1), the water is deionized water; the ratio of the carbon source to the deionized water is 1:3-5.

[0017] Preferably, in step 2), the nitrogen source is ethylenediamine; the solid-liquid ratio of the carbon source to the nitrogen source is 1:0.75-2; preferably, the solid-liquid ratio of the carbon source to the nitrogen source is 1:1.5.

[0018] Preferably, in step 3), the mixed solution is heated to 100° C. for the first time and maintained at this temperature for 10 to 40 minutes; more preferably, the temperature is maintained at the boiling temperature of water for 30 minutes.

[0019] Preferably, in step 4), after the water is completely evaporated, the set temperature of the heating reaction is changed to 140° C., and the heating reaction is further carried out for 10 to 20 minutes; preferably, the heating reaction time is 20 minutes.

[0020] In order to solve the above second technical problem, the technical solution adopted by the invention is as follows:

[0021] An application of carbonized polymer nanodots in chemiluminescence detection comprises the following steps:

[0022] 11) dissolving 45-55 mg of carbonized polymer nanodots in 45-55 ml of deionized water to obtain solution I;

[0023] 12) Using a pipette, transfer 45-55 mL of dibutyl phthalate into a beaker, and then weigh 8-12 g of bisoxalate using an electronic balance and pour it into the beaker to fully dissolve it, thereby obtaining a solution II;

[0024] 13) Use a pipette to transfer 23-27 mL of 30 wt% hydrogen peroxide and 23-27 mL of tert-butyl alcohol into a beaker, mix thoroughly, and use an electronic balance to weigh 14-16 g of anhydrous sodium acetate and dissolve it in the mixed solution to obtain solution III;

[0025] 14) Mixing solution I, solution II and solution III to obtain a bisoxalate / carbonized polymer nanodot chemiluminescent reaction solution.

[0026] Preferably, in step 14), the volume ratio of solution I:solution II:solution III is 4-16:3-5:3-5.

[0027] More preferably, in step 14), the volume ratio of solution I:solution II:solution III is 16:4:4.

[0028] Any range described in the present invention includes the end value and any numerical value between the end values ​​and any sub-range formed by the end value or any numerical value between the end values.

[0029] Unless otherwise specified, all raw materials in the present invention can be purchased from the market, and the equipment used in the present invention can adopt conventional equipment in the relevant field or refer to the existing technology in the relevant field.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1) Traditional organic fluorescent dyes and semiconductor quantum dots are highly toxic, while the carbonized polymer nanodots of the present invention have the characteristics of biocompatibility, excellent fluorescence properties, simple preparation and environmental protection, which have certain theoretical value and application research foundation for the test range, sensitivity, safety and environmental protection of chemiluminescence detection technology in the future in the field of environmental and clinical detection.

[0032] 2) The present invention adopts a more energy-saving and environmentally friendly solvent thermal method to prepare carbonized polymer nanodots. The preparation process is simple, does not require high temperature and high pressure, and has smaller particle size, more uniform distribution, and more regular shape. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0034] Figure 1 TEM images of carbonized polymer nanodots prepared in Examples 1-5 of the present invention;

[0035] Figure 2 The fluorescence spectra of the chemiluminescent reaction solution at different reaction times when different masses of carbonized polymer nanodots are added in Examples 6-9 of the present invention;

[0036] Figure 3 TEM images of carbonized polymer nanodots prepared by maintaining different heating times at a set temperature in Comparative Examples 1-3 of the present invention;

[0037] Figure 4 The fluorescence spectra of the chemiluminescent reaction solution at different reaction times when the conventional organic fluorescent dye Rhodamine B and carbonized polymer nanodots of different masses are added to Comparative Examples 4-7 of the present invention. DETAILED DESCRIPTION

[0038] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0039] For the convenience of description, the descriptions of "first", "second", etc. in the present invention are only set for descriptive purposes and cannot be understood as indicating or suggesting their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various implementation methods can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0040] As one aspect of the present invention, a method for preparing carbonized polymer nanodots for chemiluminescence detection comprises the following steps:

[0041] 1) dissolving the carbon source in water to form a uniform solution;

[0042] 2) slowly adding a nitrogen source to the above uniform solution to form a mixed solution;

[0043] 3) heating the obtained mixed solution to react;

[0044] 4) After the water is completely evaporated, the set temperature of the heating reaction is changed to transform it from a yellow liquid to a dark brown soft semi-solid;

[0045] 5) cooling the dark brown soft semi-solid to room temperature and grinding it into powder to obtain carbonized polymer nanodots;

[0046] Step 3) and step 4) are both carried out under ambient pressure.

[0047] In certain embodiments of the present invention, in step 1), the carbon source is citric acid.

[0048] In certain embodiments of the present invention, in step 1), the water is deionized water; and the ratio of the carbon source to the deionized water is 1:3-5.

[0049] In certain embodiments of the present invention, in step 2), the nitrogen source is ethylenediamine; the solid-liquid ratio of the carbon source to the nitrogen source is 1:0.75-2; preferably, the solid-liquid ratio of the carbon source to the nitrogen source is 1:1.5.

[0050] In certain embodiments of the present invention, in step 3), the mixed solution is heated to 100° C. for the first time and maintained at this temperature for 10 to 40 minutes; more preferably, the temperature is maintained at the boiling temperature of water for 30 minutes.

[0051] In certain embodiments of the present invention, in step 4), after the water is completely evaporated, the set temperature of the heating reaction is changed to 140° C., and the heating reaction is further carried out for 10 to 20 minutes; preferably, the heating reaction time is 20 minutes.

[0052] As another aspect of the present invention, an application of carbonized polymer nanodots in chemiluminescence detection comprises the following steps:

[0053] 11) dissolving 45-55 mg of carbonized polymer nanodots in 45-55 ml of deionized water to obtain solution I;

[0054] 12) Using a pipette, transfer 45-55 mL of dibutyl phthalate into a beaker, and then weigh 8-12 g of bisoxalate using an electronic balance and pour it into the beaker to fully dissolve it, thereby obtaining a solution II;

[0055] 13) Use a pipette to transfer 23-27 mL of 30 wt% hydrogen peroxide and 23-27 mL of tert-butyl alcohol into a beaker, mix thoroughly, and use an electronic balance to weigh 14-16 g of anhydrous sodium acetate and dissolve it in the mixed solution to obtain solution III;

[0056] 14) Mixing solution I, solution II and solution III to obtain a bisoxalate / carbonized polymer nanodot chemiluminescent reaction solution.

[0057] In certain embodiments of the present invention, in step 14), the volume ratio of solution I:solution II:solution III is 4-16:3-5:3-5.

[0058] More preferably, in step 14), the volume ratio of solution I:solution II:solution III is 16:4:4.

[0059] Example 1

[0060] A method for preparing carbonized polymer nanodots for chemiluminescence detection comprises the following steps:

[0061] 1) Dissolve 6 g of citric acid in 18 mL of deionized water in a 50 mL beaker;

[0062] 2) Use a pipette to accurately transfer 4.5 mL of ethylenediamine, slowly add it to the beaker while stirring at room temperature, and mix thoroughly;

[0063] 3) After mixing, the mixed solution was heated at ambient pressure, the heating temperature was set to 100°C, and kept at the boiling temperature of water for 30 minutes;

[0064] 4) After the water was completely evaporated, the set temperature was changed to 140° C. and the reaction mixture was further heated for 20 min to transform it from a yellow liquid to a dark brown soft semi-solid;

[0065] 5) After the product is cooled to room temperature, the obtained dark brown solid is ground into powder to obtain carbonized polymer nanodots.

[0066] The morphology and size of carbonized polymer nanodots were observed by Hitachi HT-7700TEM transmission electron microscope at 100 kV. Figure 1 As shown in (a), the particle size range is between 7-15nm.

[0067] Example 2

[0068] Example 1 was repeated, except that the amount of ethylenediamine was adjusted to 6 mL. The specific morphology and size are as follows: Figure 1 As shown in (b), the particle size range is between 3-15nm.

[0069] Example 3

[0070] Example 1 was repeated, except that the amount of ethylenediamine was adjusted to 7.5 mL. The specific morphology and size are as follows: Figure 1 As shown in (c), the particle size range is between 3-6 nm.

[0071] Example 4

[0072] Example 1 was repeated, except that the amount of ethylenediamine was adjusted to 9 mL. The specific morphology and size are as follows: Figure 1 As shown in (d), the particle size range is between 1-2 nm.

[0073] Example 5

[0074] Example 1 was repeated, except that the amount of ethylenediamine was adjusted to 12 mL. The specific morphology and size are as follows: Figure 1 As shown in (e), the particle size range is between 3-15nm.

[0075] Example 6

[0076] An application of carbonized polymer nanodots for chemiluminescence detection comprises the following steps:

[0077] 1) Dissolve 6 g of citric acid in 18 mL of deionized water in a 50 mL beaker;

[0078] 2) Use a pipette to accurately transfer 9 mL of ethylenediamine and slowly add it to the beaker under stirring at room temperature and mix thoroughly;

[0079] 3) After mixing, the mixed solution was heated at ambient pressure, the heating temperature was set to 100°C, and kept at the boiling temperature of water for 30 minutes;

[0080] 4) After the water was completely evaporated, the set temperature was changed to 140° C. and the reaction mixture was further heated for 20 min to transform it from a yellow liquid to a dark brown soft semi-solid;

[0081] 5) After the product is cooled to room temperature, the obtained dark brown solid is ground into powder to obtain carbonized polymer nanodots.

[0082] 6) dissolving 50 mg of carbonized polymer nanodots in 50 ml of deionized water to obtain solution I;

[0083] 7) Using a pipette, 50 mL of dibutyl phthalate was transferred into a beaker, and then 10 g of bisoxalate was weighed using an electronic balance and poured into the beaker to fully dissolve the bisoxalate to obtain a solution II;

[0084] 8) Use a pipette to transfer 25 mL of hydrogen peroxide (H2O2, 30%) and 25 mL of tert-butanol into a beaker. After thorough mixing, use an electronic balance to weigh 15 g of anhydrous sodium acetate and dissolve it in the mixed solution to obtain solution III.

[0085] 9) 4 mL of solution I, 4 mL of solution II and 4 mL of solution III were mixed to obtain a bisoxalate / carbonized polymer nanodot chemiluminescent reaction solution.

[0086] The changes in chemiluminescence fluorescence intensity were recorded by Cary 50Conc fluorescence spectrometer to obtain fluorescence emission spectra at different reaction times, such as Figure 2 As shown in (a), the fluorescence intensity gradually weakens with time, with obvious changes in a short period of time and then slow changes.

[0087] Example 7

[0088] Example 6 was repeated, except that the amount of solution I was adjusted to 8 mL. The fluorescence emission spectra at different reaction times are shown in FIG. Figure 2 As shown in (b), the fluorescence intensity decreases over time, but the rate of decrease is slow and uniform.

[0089] Example 8

[0090] Example 6 was repeated, except that the amount of solution I was adjusted to 12 mL. The fluorescence emission spectra at different reaction times are shown in FIG. Figure 2 As shown in (c), the fluorescence intensity decreases over time, but the rate of decrease is slow and uniform.

[0091] Example 9

[0092] Example 6 was repeated, except that the amount of solution I was adjusted to 16 mL. The fluorescence emission spectra at different reaction times are shown in FIG. Figure 2 As shown in (d), the fluorescence intensity decreases over time, but the rate of decrease is slow and uniform.

[0093] Comparative Example 1

[0094] The difference between this comparative example and Example 4 is that after mixing, the mixed solution is heated under ambient pressure, the heating temperature is set to 100°C, and kept at the boiling temperature of water for 10 minutes. The other methods are the same as those of Example 4. The specific morphology and size are as follows: Figure 3 As shown in (a), the particle size ranges from 10 to 60 nm.

[0095] This shows that there are spheres with larger particle sizes at this time, and the polymerization and carbonization reaction has not been completed.

[0096] Comparative Example 2

[0097] The difference between this comparative example and Example 4 is that after mixing, the mixed solution is heated under ambient pressure, the heating temperature is set to 100°C, and kept at the boiling temperature of water for 20 minutes. The other methods are the same as those of Example 4. The specific morphology and size are as follows: Figure 3 As shown in (b), the particle size ranges from 10 to 30 nm.

[0098] This shows that there are spheres with larger particle sizes at this time, and the polymerization and carbonization reaction has not been completed.

[0099] Comparative Example 3

[0100] The difference between this comparative example and Example 4 is that after the water is completely evaporated, the set temperature is changed to 140°C, and the reaction mixture is further heated for 10 minutes. The other methods are the same as those in Example 4. The specific morphology and size are as follows Figure 3 As shown in (c), the particle size range is between 10 and 30 nm.

[0101] This shows that there are spheres with larger particle sizes at this time, and the polymerization and carbonization reaction has not been completed.

[0102] Comparative Example 4

[0103] The difference between this comparative example and Example 6 is that the traditional organic fluorescent dye Rhodamine B is added during the preparation of the bisoxalate / carbonized polymer nanodot chemiluminescent reaction solution. The other methods are the same as those of Example 6. The fluorescence emission spectra at different reaction times are as follows: Figure 4 As shown in (a), the fluorescence intensity decreases over time, but the rate of decrease is not uniform.

[0104] It can be seen that when traditional organic fluorescent dyes are replaced with carbonized polymer nanodots in chemiluminescence detection, the stability of chemiluminescence is better.

[0105] Comparative Example 5

[0106] The difference between this comparative example and Example 7 is that the traditional organic fluorescent dye Rhodamine B is added during the preparation of the bisoxalate / carbonized polymer nanodot chemiluminescent reaction solution. The other methods are the same as those of Example 7. The fluorescence emission spectra at different reaction times are as shown in FIG. Figure 4 As shown in (b), the fluorescence intensity decreases over time, but the rate of decrease is fast and uneven.

[0107] It can be seen that when traditional organic fluorescent dyes are replaced with carbonized polymer nanodots in chemiluminescence detection, the stability of chemiluminescence is better.

[0108] Comparative Example 6

[0109] The difference between this comparative example and Example 8 is that the traditional organic fluorescent dye Rhodamine B is added during the preparation of the bisoxalate / carbonized polymer nanodot chemiluminescent reaction solution. The other methods are the same as those of Example 8. The fluorescence emission spectra at different reaction times are as shown in FIG. Figure 4 As shown in (c), the fluorescence intensity decreases over time, but the rate of decrease is fast and uneven.

[0110] It can be seen that when traditional organic fluorescent dyes are replaced with carbonized polymer nanodots in chemiluminescence detection, the stability of chemiluminescence is better.

[0111] Comparative Example 7

[0112] The difference between this comparative example and Example 9 is that the traditional organic fluorescent dye Rhodamine B is added during the preparation of the bisoxalate / carbonized polymer nanodot chemiluminescent reaction solution. The other methods are the same as those of Example 9. The fluorescence emission spectra at different reaction times are as shown in FIG. Figure 4 As shown in (d), the fluorescence intensity decreases over time, but the rate of decrease is fast and uneven.

[0113] It can be seen that when traditional organic fluorescent dyes are replaced with carbonized polymer nanodots in chemiluminescence detection, the stability of chemiluminescence is better.

[0114] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all implementation methods here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for preparing carbonized polymer nanodots for chemiluminescence detection, characterized in that: The steps include: 1) dissolving the carbon source in water to form a uniform solution; 2) slowly adding a nitrogen source to the above uniform solution to form a mixed solution; 3) heating the obtained mixed solution to react; 4) After the water is completely evaporated, the set temperature of the heating reaction is changed to transform it from a yellow liquid to a dark brown soft semi-solid; 5) cooling the dark brown soft semi-solid to room temperature and grinding it into powder to obtain carbonized polymer nanodots; Step 3) and step 4) are both carried out under ambient pressure.

2. The method for preparing carbonized polymer nanodots for chemiluminescence detection according to claim 1, characterized in that: In step 1), the carbon source is citric acid.

3. The method for preparing carbonized polymer nanodots for chemiluminescence detection according to claim 1, characterized in that: In step 1), the water is deionized water; the ratio of the carbon source to the deionized water is 1:3-5.

4. The method for preparing carbonized polymer nanodots for chemiluminescence detection according to claim 1, characterized in that: In step 2), the nitrogen source is ethylenediamine; the solid-liquid ratio of the carbon source to the nitrogen source is 1:0.75-2; preferably, the solid-liquid ratio of the carbon source to the nitrogen source is 1:1.

5.

5. The method for preparing carbonized polymer nanodots for chemiluminescence detection according to claim 1, characterized in that: In step 3), the mixed solution is heated to 100° C. for the first time and maintained at this temperature for 10 to 40 minutes; more preferably, the mixture is maintained at the boiling temperature of water for 30 minutes.

6. The method for preparing carbonized polymer nanodots for chemiluminescence detection according to claim 1, characterized in that: In step 4), after the water is completely evaporated, the set temperature of the heating reaction is changed to 140° C., and the heating reaction is further carried out for 10 to 20 minutes; preferably, the heating reaction time is 20 minutes.

7. Use of any carbonized polymer nanodots prepared as claimed in claims 1 to 6 in chemiluminescence detection, characterized in that: The steps include: 11) dissolving 45-55 mg of carbonized polymer nanodots in 45-55 ml of deionized water to obtain solution I; 12) Using a pipette, transfer 45-55 mL of dibutyl phthalate into a beaker, and then weigh 8-12 g of bisoxalate using an electronic balance and pour it into the beaker to fully dissolve it, thereby obtaining a solution II; 13) Use a pipette to transfer 23-27 mL of 30 wt% hydrogen peroxide and 23-27 mL of tert-butyl alcohol into a beaker, mix thoroughly, and use an electronic balance to weigh 14-16 g of anhydrous sodium acetate and dissolve it in the mixed solution to obtain solution III; 14) Mixing solution I, solution II and solution III to obtain a bisoxalate / carbonized polymer nanodot chemiluminescent reaction solution.

8. The application according to claim 7, characterized in that: In step 14), the volume ratio of solution I:solution II:solution III is 4-16:3-5:3-5.

9. The use according to claim 8, characterized in that: The volume ratio of solution I: solution II: solution III is 16:4:4.

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