CN102617779B - 2,3,4,5-tetraphenylsilole-based water-soluble polymer and its preparation method and use - Google Patents

Abstract

The invention discloses a 2,3,4,5-tetraphenylsilole-based water-soluble polymer and its preparation method and use. The 2,3,4,5-tetraphenylsilole-based water-soluble polymer has a general structural formula shown in the formula I. In invention, 2,3,4,5-tetraphenylsilole comonomers and water-soluble monomers undergo a free-radical copolymerization reaction to be synthesized into a series of water-soluble polymers having side chain structures formed of 2,3,4,5-tetraphenylsilole. The 2,3,4,5-tetraphenylsilole-based water-soluble polymer provided by the invention has good luminescence properties, can be dissolved in water, has good fluorescence quenching or fluorescence enhancement effects on certain specific groups or substances, and can realize chemical/biological detection in an aqueous solution. Therefore, the 2,3,4,5-tetraphenylsilole-based water-soluble polymer is a novel chemical/biological detection material.

Description

Water-soluble polymer based on 2,3,4,5-tetraphenylsilacyclopentadiene and its preparation method and application

technical field

The invention relates to a water-soluble polymer based on 2,3,4,5-tetraphenylsilacyclopentadiene, a preparation method and application thereof. the

Background technique

Ordinary fluorescent substances only have fluorescence in organic solvents, and fluorescence quenching occurs in the aggregated state. Therefore, organic fluorescent substances used in chemical/biological detection can only be carried out in organic solvents, and the solubility of more detected substances in organic solvents is not ideal, and most of the active substances are in organic solvents. It will lose its activity and affect the reliability of the detection results, so the development of water-soluble detection system has become the focus of research. the

Water-soluble organic fluorescent small molecules tend to aggregate in aqueous solution due to the hydrophobicity of the chromophore, resulting in low fluorescence intensity. Therefore, there are also many problems to be solved, such as weak luminescence intensity and low detection sensitivity. Water-soluble fluorescent quantum dots and water-soluble conjugated polymers developed in recent years have attracted widespread attention. the

Water-soluble quantum dots are usually inorganic metal compound nanoparticles that are endowed with water solubility through surface modification. However, such compounds contain heavy metals, which cause certain pollution to the environment, and when used for the detection of biologically active biomolecules, the problem of activity loss will also be caused. The water-soluble conjugated polymer is an amphiphilic molecule, the main chain is a rigid conjugated ring, and the side chain is a water-soluble group. For such linear molecules with a rigid structure, the result of the balance between the hydrophobic interaction of the conjugated main chain and the hydrophilic interaction of the flexible side chain is the formation of an aggregated state in aqueous solution, which also causes fluorescence weakening, detection sensitivity, and When used for biomolecular detection, it cannot fully interact with biomolecules. the

2,3,4,5-Tetraphenylsilacyclopentadiene is a special class of small organic fluorescent molecules. They are non-fluorescent in organic solvents but highly fluorescent in aggregated and solid states. This opens up the possibility of their use in chemical/biological detection of aqueous systems. High fluorescence intensity endows these compounds with high detection sensitivity. However, in the aqueous detection systems based on small molecules or polymers of such compounds that have been developed so far, good dispersion is still a problem to be solved. the

Contents of the invention

The object of the present invention is to provide a water-soluble polymer based on 2,3,4,5-tetraphenylsilacyclopentadiene, its preparation method and application. the

The water-soluble polymer based on 2,3,4,5-tetraphenylsilacyclopentadiene provided by the invention is a polymer shown in formula I,

(formula I)

In the general structural formula of formula I, _R1 is an alkyl, cyclohexyl, phenyl or p-methylphenyl group with 1-6 carbon atoms, preferably methyl; _R2 is hydroxyl, amino or isopropylamine R is a hydrogen atom or a methyl group; m and n are _both integers of 1-200, m is preferably an integer of 1-50, more preferably 1, and n is preferably an integer of 1-50, specifically 48-144 , an integer of 53-144 or 48-53.

The method for preparing the above-mentioned polymer provided by the present invention comprises the following steps: in the presence of a free radical initiator, carry out a copolymerization reaction of the compound represented by formula IV with a water-soluble monomer in an organic solvent, and obtain the polymerized things. the

In the above method, the organic solvent is selected from at least one of toluene, tetrahydrofuran and dichloromethane, preferably tetrahydrofuran; the free radical initiator is azobisisobutyronitrile or benzoyl peroxide; the water-soluble The monomer is acrylic acid, methacrylic acid, acrylamide or N-isopropylacrylamide; the molar ratio of the free radical initiator, the compound shown in formula IV and the water-soluble monomer is 0.1-0.3:1-1.1:1 -100, specifically 0.2-0.3:1:80-100, preferably 0.2:1:40; in the copolymerization step, the temperature is 20-80°C, preferably 70°C; the time is 1-24 hours, preferably 24 Hour. the

Wherein, the compound represented by the formula IV (that is, the monomer containing 2,3,4,5-tetraphenylsilacyclopentadiene) also belongs to the protection scope of the present invention. the

The general structural formula of the compound shown in this formula IV is as follows:

(Formula IV)

In the general structural formula of formula IV, R ₁ is an alkyl group with 1-6 carbon atoms, cyclohexyl group, phenyl group or p-methylphenyl group, preferably methyl group.

The method for preparing the compound shown in the above formula IV provided by the present invention comprises the following steps: in the presence of dicyclohexylcarbodiimide and 4-dimethylaminopyridine, the compound shown in formula III is mixed with acrylic acid in an organic solvent The reaction is carried out, and the compound is obtained after the reaction is completed. the

In the above method, the organic solvent is selected from at least one of toluene, tetrahydrofuran and dichloromethane, preferably toluene; the compound shown in the dicyclohexylcarbodiimide, 4-dimethylaminopyridine, formula III and acrylic acid The molar ratio is 1-3: 0.01-0.1: 1-1.2: 1-10, preferably 1.5: 0.06: 1: 4; in the reaction step, the temperature is 20-80°C, preferably 25°C; the time is 1- 20 hours, preferably 14 hours. the

Wherein, the compound represented by the formula III (ie 2,3,4,5-tetraphenylsilacyclopentadiene substituted with amino group) also belongs to the protection scope of the present invention. the

The general structural formula of the compound shown in this formula III is as follows:

(formula III)

In the general structural formula of formula III, R ₁ is an alkyl group with 1-6 carbon atoms, cyclohexyl, phenyl or p-methylphenyl, preferably methyl.

The method for preparing the compound shown in the above formula III provided by the present invention comprises the following steps: reacting the compound shown in formula II with p-alkynyl aniline in an organic solvent in the presence of chloroplatinic acid, and the reaction is completed to obtain the Compound shown in formula III;

(formula II)

In the general structural formula of the formula II, R ₁ is an alkyl, cyclohexyl, phenyl or p-methylphenyl group with 1-6 carbon atoms, preferably methyl; the organic solvent is selected from toluene, tetrahydrofuran and dihydrofuran At least one of methyl chloride, preferably toluene; the molar ratio of the chloroplatinic acid, the compound represented by formula II and p-alkynyl aniline is 1×10 ^-6 -5×10 ^-6 : 1-1.1 : 1-1.1 , preferably 2×10 ⁻⁶ : 1:1; in the reaction step, the temperature is 20-110° C., preferably 110° C.; the time is 1-24 hours, preferably 24 hours.

In addition, the application of the polymer represented by formula I provided by the present invention in chemical detection or biological detection methods also belongs to the protection scope of the present invention. The method for the polymer represented by the formula I protected by the present invention in chemical or biological detection can be specifically: the water-soluble polymer provided by the present invention is made into a 6mg/L dilute solution with deionized water; The dilute solution of the substance to be detected is added to 10 ml of the dilute polymer solution through a pipette gun, and the fluorescence intensity of the mixed solution of the polymer and the substance to be detected and the fluorescence intensity of the dilute polymer solution itself are measured. the

The invention adopts a simple free radical copolymerization method, introduces 2,3,4,5-tetraphenylsilacyclopentadiene into the side chain of the water-soluble polymer, and endows the polymer with good dispersibility in aqueous solution and high fluorescence intensity. Structural characterization of the polymer and determination of its luminescent properties show that the polymer has good luminescent properties and strong luminous efficiency, which will greatly improve the sensitivity of detection and provide a basis for the detection of trace substances; and the polymer It can be dissolved in water and has a good fluorescence quenching or fluorescence enhancement effect on some specific groups or substances. The realization of polymer water solubility can greatly improve the detection range, from small molecular inorganic metal ions to macromolecules Proteins or DNA fragments can be detected in aqueous solution. And this kind of polymer main chain has a large degree of conformational freedom, which is conducive to fully interacting with the substance to be detected. Therefore, the water-soluble polymer based on 2,3,4,5-tetraphenylsilacyclopentadiene provided by the present invention will have a very wide application prospect in chemical/biological detection. the

Description of drawings

Fig. 1 is the fluorescence spectrum of the polymer aqueous solution synthesized in Example 1 under the excitation of 365nm wavelength. the

Fig. 2 is the fluorescence quenching effect of the copper (II) ion concentration on the aqueous polymer solution in Example 6. the

Figure 3 is the relationship between the fluorescence intensity of the aqueous polymer solution and the concentration of bovine serum albumin in Example 7. the

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial sources unless otherwise specified. The numerical values of m and n in the structural formula of the water-soluble polymer based on 2,3,4,5-tetraphenylsilacyclopentadiene obtained in the following examples were determined according to the conventional GPC method. the

Synthesis of 1-methyl-1-(4-amino)phenyl-2,3,4,5-tetraphenylsilacyclopentadiene shown in embodiment 1, formula III

Under a nitrogen atmosphere, 240mg (0.6mmol) of 1-methyl-2,3,4,5-tetraphenylsilacyclopentadiene was dissolved in 20ml of toluene, and 70mg (0.6mmol) of p-alkynylaniline and 20uL isopropanol solution of chloroplatinic acid (the molar amount of chloroplatinic acid in this solution is 1.2× ^10-6 mmol), heated at 110°C for 24 hours, the reaction was completed, after the reaction solution was cooled, it was concentrated on a rotary evaporator and used The product was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate volume ratio 3/1), and the product was further recrystallized with a mixed solvent of n-hexane/tetrahydrofuran to obtain 259 mg of a yellow-green solid with a yield of 84%.

The NMR and high-resolution mass spectrometry detection results of the product are as follows:

¹ HNMR (400MHz, acetone-d ₆ , ppm) δ7.24 (d, 2H, J=8), 7.10-7.00 (m, 17H), 6.89-6.86 (m, 4H), 6.62 (d, 2H, J =8), 6.30(d, IH, J=19), 4.90(s, 2H), 0.61(s, 3H). ¹³ CNMR (400MHz, acetone-d ₆ , ppm) δ155.612, 150.352, 149.815, 141.731 ，140,625，140.028，130.687，129.752，128.912，128.694，128.550，128.318，127.845，127.122，126.527，126.435，114.818，114.693，-5.886.HRMS(EI)：Calcd for C ₃₇ H ₃₁ SiN：517.2226.Found：517.2231 .

From the above detection results, it can be seen that the yellow-green solid substance is amino-substituted 2,3,4,5-tetraphenylsilacyclopentadiene shown in formula III provided by the present invention, and its structural formula is shown in formula V:

(Formula V)

Synthesis of the comonomer containing 2,3,4,5-tetraphenylsilacyclopentadiene shown in embodiment 2, formula IV

934.7mg (1.81mmol) of 1-methyl-1-(4-amino)phenyl-2,3,4,5-tetraphenylsilacyclopentadiene prepared in Example 1 was dissolved in 20ml of toluene, Add 579.2mg (2.8mmol) of dicyclohexylcarbodiimide, 13.5mg (0.11mmol) of 4-dimethylaminopyridine and 0.5mL (7.3mmol) of acrylic acid in sequence, and stir at room temperature for 14 hours at 25°C to react, and the reaction is complete After filtering the insoluble matter, the filtrate was concentrated on a rotary evaporator and separated and purified by silica gel column chromatography (the volume ratio of petroleum ether/ethyl acetate was 3/1 for washing), and the product was further recrystallized with ethanol to obtain 663.8 mg of yellow-green crystals , the yield was 65%. the

The NMR and high-resolution mass spectrometry detection results of the product are as follows:

¹ HNMR (400MHz, acetone-d ₆ , ppm) δ9.38(s, 1H), 7.73(d, 2H, J=8), 7.48(d, 2H, J=8), 7.19(d, 1H, J =19), 7.11～7.03(m, 16H), 6.89～6.84(m, 4H), 6.63(d, 1H, J=19), 6.44(m, 1H), 6.34(d, 1H), 5.71(d , 1H), 0.65(s, 3H); ¹³ CNMR (400MHz, acetone-d ₆ , ppm) δ163.993, 156.055, 148.739, 141.424, 140.697, 140.527, 140.004, 134.357, 132.728, 130.7701, 129.88 , 128.183, 127.229, 126.566, 120.768, 120.254, -6.009; HRMS (EI): Calcd for C ₄₀ H ₃₃ NOSi: 571.2331. Found: 571.2337.

From the above detection results, it can be seen that the yellow-green crystal is a compound containing 2,3,4,5-tetraphenylsilacyclopentadiene shown in formula IV provided by the present invention, and its structural formula is shown in formula VI:

(Formula VI)

Embodiment 3, based on 2,3,4, the synthesis of the water-soluble polymer of 5-tetraphenylsilacyclopentadiene

Under a nitrogen atmosphere, 28.6 mg (0.05 mmol) of the comonomer compound containing 2,3,4,5-tetraphenylsilacyclopentadiene prepared in Example 2 was dissolved in 10 mL of nitrogen bubbled for 1 hour in advance. In tetrahydrofuran, then add 137μL (2mmol) of water-soluble monomer acrylic acid, stir well and then add 1.6mg (0.01mmol) of free radical initiator azobisisobutyronitrile, and the reaction solution is stirred and reacted at 70°C for 24 hours to obtain polymerization The polymer solution was settled in n-hexane and filtered, the filter cake was washed several times with n-hexane, and the obtained solid was vacuum-dried at 60° C. to obtain 124.3 mg of yellow-green solid powder with a yield of 72%. the

The detection result of this product is as follows:

IR: 3190cm ^-1 , 2955cm ^-1 , 1714cm ^-1 , 1557cm ^-1 , 1451cm ^-1 , 1407cm ^-1 ; 1254cm ^-1 , 1179cm ^-1 , 803cm ^-1 , 700cm ^-1 . ¹ HNMR (400MHz, acetone-d ₆ , ppm) δ12.34(s), 8.0～6.64(m), 2.22(s), 1.76～1.17(m), 0.61(s). The number average molecular weight is 1100, and the molecular weight distribution width is 3.18.

It can be seen from the above that the product is a water-soluble polymer based on 2,3,4,5-tetraphenylsilacyclopentadiene represented by formula I provided by the present invention, wherein m and n are 1 and 53, respectively. the

Synthesis of the water-soluble polymer based on 2,3,4,5-tetraphenylsilacyclopentadiene shown in embodiment 4, formula I

Under a nitrogen atmosphere, 28.6 mg (0.05 mmol) of the comonomer compound containing 2,3,4,5-tetraphenylsilacyclopentadiene prepared in Example 2 was dissolved in 10 ml of nitrogen bubbled for 1 hour in advance. Add 342 μL (5 mmol) of water-soluble monomer acrylic acid to tetrahydrofuran, stir the mixture evenly, then add 1.6 mg (0.01 mmol) of free radical initiator azobisisobutyronitrile, and stir the reaction solution at 70°C for 24 hours to obtain The polymer solution was filtered after sedimentation in n-hexane, the filter cake was washed several times with n-hexane, and the obtained solid was vacuum-dried at 60° C. to obtain 320.0 mg of yellow-green solid powder with a yield of 82%. the

The detection result of this product is as follows:

IR: 3190cm ^-1 , 2955cm ^-1 , 1714cm ^-1 , 1557cm ^-1 , 1451cm ^-1 , 1407cm ^-1 ; 1254cm ^-1 , 1179cm ^-1 , 803cm ^-1 , 700cm ^-1 . ¹ HNMR (400MHz, acetone-d ₆ , ppm) δ12.25(s), 8.0～6.64(m), 2.22(s), 1.76～1.17(m), 0.61(s). The number average molecular weight is 1100, and the molecular weight distribution width is 1.81.

It can be seen from the above that the product is a water-soluble polymer based on 2,3,4,5-tetraphenylsilacyclopentadiene represented by formula I provided by the present invention, wherein m and n are 1 and 144, respectively. the

Synthesis of the water-soluble polymer based on 2,3,4,5-tetraphenylsilacyclopentadiene shown in embodiment 5, formula I

Under a nitrogen atmosphere, 28.6 mg (0.05 mmol) of the comonomer compound containing 2,3,4,5-tetraphenylsilacyclopentadiene prepared in Example 2 was dissolved in 10 ml of nitrogen bubbled for 1 hour in advance. In tetrahydrofuran, then add 452.6mg (4mmol) water-soluble monomer N-isopropylacrylamide, stir evenly, then add 2.5mg (0.015mmol) free radical initiator azobisisobutyronitrile, and the reaction solution is heated at 70°C Stirring for 24 hours to obtain a polymer solution, the polymer solution was settled in n-hexane and filtered, the filter cake was washed several times with n-hexane, and the resulting solid was vacuum-dried at 80°C to obtain 384.7 mg of a yellow-green solid powder. The yield was 80%. the

The detection result of this product is as follows:

IR: 3306cm ^-1 , 3077cm ^{-1 , 2973cm -1 , 2934cm -1 , 2876cm -1 , 1649cm -1 , 1544cm -1 , 1460cm -1 , 1387cm -1 ; 1368cm -1 ,} 1173cm ^-1 , 1131cm ^-1 . ¹ HNMR (400MHz, acetone-d ₆ , ppm) δ8.0～6.64(m), 3.85(s), 2.34～1.70(m), 1.70～1.18(m), 1.04(m), 0.61(s). The number average molecular weight was 2200, and the molecular weight distribution width was 1.68.

It can be known from the above that the product is a water-soluble polymer based on 2,3,4,5-tetraphenylsilacyclopentadiene represented by formula I provided by the present invention, wherein m and n are 1 and 48, respectively. the

Example 6

The water-soluble polymer prepared in Example 3 was dissolved in deionized water to prepare a dilute solution with a concentration of 6 mg/L. Add 1.7×10 ^-8 mol/L, 3.4×10 ^-8 mol/L, 5.1×10 ^-8 mol/L, 6.8×10 ^-8 mol/L, 10.2×10 ^-8 respectively to 10ml polymer dilute solution mol/L, 13.6×10 ^-8 mol/L, 17×10 ^-8 mol/L, 20.4×10 ^-8 mol/L, 23.8×10 ^-8 mol/L copper bromide aqueous solution, compare the whole mixed solution system The fluorescence intensity I of the polymer dilute solution itself and the relative size of the fluorescence intensity I ₀ of the polymer dilute solution, the quenching effect of copper (II) ions on the fluorescence of the polymer, the results are shown in Figure 2. It can be seen from Figure 2 that the polymer has high sensitivity, and the detection of copper (II) ion concentration can be as low as 10 ^-8 mol/L.

Example 7

The polymer prepared in Example 3 was dissolved in deionized water to prepare a dilute solution with a concentration of 6 mg/L. Add different concentrations (0uM, 0.08uM, 0.16uM, 0.24uM, 0.32uM, 0.4uM, 0.48uM, 0.56uM and 0.64uM) of the phosphate buffer solution (pH Values are all 7.4), study the relationship between the fluorescence intensity of the whole mixed solution system and the bovine serum albumin concentration, as shown in Figure 3. It can be seen from the figure that the fluorescence intensity of the mixed system increases with the increase of bovine serum albumin concentration (0-0.48 μM), which can be used to detect the concentration of biomolecules. the

Claims (18)

1. polymer shown in formula I, In the general structural formula of formula I, _R1 is an alkyl, cyclohexyl, phenyl or p-methylphenyl group with 1-6 carbon atoms; _R2 is hydroxyl, amino or isopropylamino; _R3 is a hydrogen atom or a methyl group; m and n are both integers of 1-200. 2. The polymer according to claim 1, characterized in that: in the general structural formula of formula I, R ₁ is a methyl group; R ₂ is a hydroxyl group; m and n are all integers of 1-50. 3. A method for preparing the described polymer of claim 1 or 2, comprising the steps of: under the condition that a radical initiator exists, the compound shown in formula IV is carried out copolymerization reaction with water-soluble monomer in organic solvent, After the reaction is completed, the polymer described in claim 1 or 2 is obtained;

In the general structural formula of formula IV, R ₁ is an alkyl group, cyclohexyl group, phenyl group or p-methylphenyl group with 1-6 carbon atoms; The water-soluble monomer is acrylic acid, methacrylic acid, acrylamide or N-isopropylacrylamide. 4. The method according to claim 3, characterized in that: in the general structural formula of the formula IV, R ₁ is a methyl group. 5. according to the described method of claim 3 or 4, it is characterized in that: described organic solvent is selected from at least one in toluene, THF and methylene dichloride; Described radical initiator is azobisisobutyronitrile or Benzoyl peroxide; the molar ratio of the free radical initiator, the compound shown in the formula IV and the water-soluble monomer is 0.1-0.3:1-1.1:1-100; in the copolymerization reaction step, the temperature is 20-80°C; time is 1-24 hours. 6. The method according to claim 5, characterized in that: the organic solvent is selected from tetrahydrofuran; the molar ratio of the radical initiator, the compound shown in the formula IV and the water-soluble monomer is 0.2:1: 40; in the copolymerization reaction step, the temperature is 70° C.; the time is 24 hours. 7. The use of the polymer according to claim 1 or 2 in chemical detection or biological detection methods. 8. The compound shown in formula IV;

In the general structural formula of formula IV, R ₁ is an alkyl group with 1-6 carbon atoms, cyclohexyl, phenyl or p-methylphenyl. 9. The compound according to claim 8, characterized in that: in the general structural formula of formula IV, R ₁ is a methyl group. 10. A method for preparing the compound described in claim 8 or 9, comprising the steps of: under the condition that dicyclohexylcarbodiimide and 4-dimethylaminopyridine exist, the compound shown in formula III is dissolved in an organic solvent Reacting with acrylic acid, the reaction is completed to obtain the compound described in claim 8 or 9;

In the general structural formula of formula III, R ₁ is an alkyl group with 1-6 carbon atoms, cyclohexyl, phenyl or p-methylphenyl. 11. The method according to claim 10, characterized in that: in the general structural formula of the formula III, R ₁ is a methyl group. 12. The method according to claim 10 or 11, characterized in that: the organic solvent is selected from at least one of toluene, THF and methylene chloride; The molar ratio of the dicyclohexylcarbodiimide, 4-dimethylaminopyridine, the compound represented by the formula III and acrylic acid is 1-3:0.01-0.1:1-1.2:1-10; In the reaction step, the temperature is 20-80° C.; the time is 1-20 hours. 13. The method according to claim 12, characterized in that: the organic solvent is selected from toluene; The molar ratio of the dicyclohexylcarbodiimide, 4-dimethylaminopyridine, the compound represented by the formula III and acrylic acid is 1.5:0.06:1:4; In the reaction step, the temperature is 25° C.; the time is 14 hours. 14. The compound shown in formula III,

In the general structural formula of formula III, R ₁ is an alkyl group with 1-6 carbon atoms, cyclohexyl, phenyl or p-methylphenyl. 15. The compound according to claim 14, characterized in that: in the general structural formula of formula III, R ₁ is a methyl group. 16. A method for preparing the compound described in claim 14 or 15, comprising the steps of: reacting the compound shown in formula II with p-alkynyl aniline in an organic solvent in the presence of chloroplatinic acid, and obtaining The compound of claim 14 or 15;

In the general structural formula of formula II, R ₁ is an alkyl group with 1-6 carbon atoms, cyclohexyl, phenyl or p-methylphenyl. 17. the method according to claim 16 is characterized in that: in described formula II structure general formula, R Be _methyl ; Described organic solvent is selected from at least one in toluene, tetrahydrofuran (THF) and methylene dichloride; The molar ratio of chloroplatinic acid, the compound shown in formula II and p-alkynyl aniline is 1×10 ^-6 -5×10 ^-6 : 1-1.1: 1-1.1; in the reaction step, the temperature is 20-110 °C; time ranges from 1 to 24 hours. 18. The method according to claim 17, characterized in that: the organic solvent is toluene; the molar ratio of the chloroplatinic acid, the compound shown in formula II and p-alkynyl aniline is 2×10 ⁻⁶ : 1: 1; in the reaction step, the temperature is 110° C.; the time is 24 hours.

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