Aqueous two-phase system for detecting aromatic amino acid and selective response of aqueous two-phase system to aromatic amino acid

Abstract

The invention discloses an aqueous two-phase system for detecting aromatic amino acid and selective response of the aqueous two-phase system to the aromatic amino acid. An S-DPED / SDS / Na2CO3 aqueous two-phase system (ATPS) is constructed for the first time, the upper phase is composed of S-DPED and SDS, and the lower phase is a Na2CO3 solution. The ATPS constructed by the invention can be used for identifying three aromatic amino acids, namely phenylalanine, tryptophan and tyrosine. By adding three aromatic amino acids into ATPS, it is found that the difference of minimum concentrations (namely critical concentrations) required for converting the ATPS into a single phase is large. In addition, when aliphatic amino acids are added to the ATPS, the system does not become a single phase. When the concentration of the three aromatic amino acids is lower than the critical concentration, the distribution difference of the three aromatic amino acids in the upper and lower phases of ATPS is significant, which indicates that the aqueous two-phase system has selective response to the three aromatic amino acids, and detection and identification of the aromatic amino acids are realized.

Description

Technical Field

[0001] The invention belongs to the field of amino acid detection, and in particular relates to a two-aqueous phase system for detecting aromatic amino acids and a selective response thereof to the aromatic amino acids. Background Art

[0002] Amino acids containing benzene rings in their molecular structures are called aromatic amino acids (AAAs), including phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). Aromatic amino acids are of great significance in protein chemistry, clinical diagnosis, and research.

[0003] There are many methods for detecting aromatic amino acids, such as high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS), capillary electrophoresis (CE), gas chromatography (GC), amino acid analyzer, fluorescence detection, ultra-high performance liquid chromatography (UPLC), etc. However, these detection methods have their limitations, such as high cost and complex operation. Aqueous two-phase system (ATPS) is a new liquid-liquid extraction technology with mild conditions and good stability, so it has attracted widespread attention. Traditional ATPS include polymer-polymer, polymer-salt, polymer-surfactant, surfactant-salt, ionic liquid-salt, alcohol-salt and other ATPS. Researchers used ionic liquid / PEG 400 / citrate ATPS (RCSSousa et al., Separation and Purification Technology, 2018, 196, 244-253), ethyl lactate / K2CO3 ATPS (I. Kamalanathan et al., Fluid Phase Equilibria, 2018, 462, 6-13) and deep eutectic mixture PDES / K2HPO4 (or K3PO4) ATPS (C. Cai et al., Food Chemistry, 2024, 430, 137029) to extract the three aromatic amino acids, and found that the three aromatic amino acids were mainly distributed in the upper phase of ATPS. When polyethylene glycol dimethyl ether 250 / choline chloride / sucrose (or fructose) ATPS (I. Sogutlu et al., RSC Advances, 2023, 13, 19674-19681) is used to extract L-arginine, L-phenylalanine and L-tyrosine, the three amino acids are mainly distributed in the lower phase of ATPS.

[0004] In summary, when the existing ATPS extracts aromatic amino acids, the extracted aromatic amino acids will be enriched in a certain phase and have no selective response to aromatic amino acids, so it cannot be used for the detection of aromatic amino acids. Summary of the invention

[0005] Purpose of the invention: In order to solve the above technical problems, the present invention aims to provide a two-phase aqueous system for detecting aromatic amino acids. The S-DPED / SDS / Na2CO3 two-phase aqueous system constructed by the present invention realizes the selective distribution of aromatic amino acids and can be used to detect and identify three aromatic amino acids. The detection method is simple in sample preparation, does not require any detection equipment, is low in cost, and solves the limitations of current amino acid detection methods.

[0006] The present invention also provides an application of a two-phase aqueous system constructed by using S-DPED / SDS / Na2CO3 in detecting and identifying aromatic amino acids.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a two-phase aqueous system for detecting aromatic amino acids, comprising S-DPED, SDS and Na2CO3, wherein the structural formula of the S-DPED is: The structural formula of SDS is:

[0008] Among them, the upper phase of the two-phase aqueous system is composed of S-DPED and SDS, and the lower phase is composed of Na2CO3.

[0009] Among them, the total concentration of S-DPED and SDS in the two-phase aqueous system is 2wt.%~10wt.%, and the concentration of Na2CO3 is 8wt.%~22wt.%, which is sufficient to ensure that it is in the two-phase region of the two-phase aqueous system, and deionized water is used as the solvent.

[0010] Furthermore, in the two-phase aqueous system, the concentration of S-DPED is 0.34wt.% to 2.96wt.%, the concentration of SDS is 1.41wt.% to 8.26wt.%, and the concentration of Na2CO3 is 11wt.% to 19wt.%.

[0011] Furthermore, in the two-phase aqueous system, n S-DPED :n SDS =2:7-4:7.

[0012] Preferably, the molar ratio of S-DPED to SDS in the aqueous two-phase system is 2:7 or 4:7. S-DPED :n SDS =2:7, the mass concentrations of S-DPED are 0.34wt% and 0.70wt.%, and the corresponding mass concentrations of SDS are 1.66wt% and 3.30wt.%. S-DPED :n SDS=4:7, the mass concentrations of S-DPED were 0.59wt% and 1.18wt.%, and the corresponding mass concentrations of SDS were 1.41wt% and 2.82wt.%.

[0013] Preferably, in the S-DPED / SDS / Na2CO3 aqueous two-phase system, the preferred concentration of S-DPED is 0.34wt.% to 1.18wt.%, the preferred concentration of SDS is 1.41wt.% to 3.30wt.%, and the preferred concentration of Na2CO3 is 12.00wt.% to 14.00wt.%.

[0014] Furthermore, the concentration of S-DPED in the two-phase aqueous system is 0.34wt.%, 0.70wt.%, 0.59wt.% or 1.18wt.%, the corresponding concentration of SDS is 1.66wt.%, 3.30wt.%, 1.41wt.% or 2.82wt.%, and the concentration of Na2CO3 is 12.00wt.% or 14.00wt.%.

[0015] Most preferably, the ATPS is 0.70wt.% S-DPED / 3.30wt.% SDS / 14.00wt.% Na2CO3, ATPS (n S-DPED :n SDS =2:7).

[0016] The method for preparing the two-phase aqueous system for detecting aromatic amino acids of the present invention comprises the steps of:

[0017] S-DPED, SDS and Na2CO3 were added to deionized water, mixed evenly and allowed to stand for equilibrium, and then centrifuged to obtain a two-phase aqueous system.

[0018] The two-aqueous phase system for detecting aromatic amino acids of the present invention is used in realizing the selective distribution of aromatic amino acids and detecting aromatic amino acids.

[0019] Wherein, the aromatic amino acid includes any one of phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr).

[0020] The application is to add aromatic amino acid solutions to a two-phase aqueous system containing S-DPED / SDS / Na2CO3, respectively, mix well, balance and centrifuge.

[0021] Wherein, the concentration of the aromatic amino acid is 0-200 μmol / g. The three aromatic amino acids, namely phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr), have different minimum concentrations (i.e., critical concentrations) required to transform the ATPS into a single phase. Thus, the three aromatic amino acids can be detected by the minimum amino acid concentration required for the ATPS to transform into a single phase. Below the critical concentration, tyrosine (Tyr) is mainly distributed in the lower phase of ATPS, tryptophan (Trp) is mainly distributed in the upper phase of ATPS, and the distribution of phenylalanine (Phe) in the upper and lower phases of ATPS is between Tyr and Trp.

[0022] The present invention uses S-DPED / SDS / Na2CO3 to construct a two-phase aqueous system, and based on the difference in critical concentrations of the three aromatic amino acids for converting the ATPS into a single phase, the detection and identification of the three aromatic amino acids are achieved.

[0023] The present invention adds aromatic amino acid solutions of different concentrations to the S-DPED / SDS / Na2CO3 aqueous two-phase system, mixes, balances, centrifuges, and observes the influence of aromatic amino acids on the phase behavior of the aqueous two-phase system, and obtains the minimum concentration of amino acids required for the system to change from two phases to a single phase after adding three aromatic amino acids respectively, wherein the critical concentration of tryptophan (Trp) is the lowest, followed by phenylalanine (Phe), and the critical concentration of tyrosine (Tyr) is the highest. Based on the significant difference in the critical concentrations of the three aromatic amino acids that cause the system to undergo phase transition, the three can be distinguished. Further, when the amino acid concentration is lower than its critical concentration, the distribution difference of the three aromatic amino acids in the upper and lower phases of the aqueous two-phase is significant, which causes the three aromatic amino acids to show different abilities to cause the system to undergo phase transition. Thus, the present invention utilizes the selective response of the aqueous two-phase system constructed by S-DPED / SDS / Na2CO3 to aromatic amino acids, and realizes the detection of aromatic amino acids. In addition, the present invention also found that when the concentration of aliphatic amino acids (including threonine, alanine, valine, lysine, glutamine, serine, glycine, methionine, aspartic acid, and arginine) is much greater than the critical concentration of the above three aromatic amino acids, ATPS will not transform into a single phase, which further demonstrates the excellent performance of the system in the detection of aromatic amino acids.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0025] 1. The present invention constructs a S-DPED / SDS / Na2CO3 two-phase aqueous system for the first time, wherein the upper phase is mainly composed of S-DPED and SDS solution, and the lower phase is mainly composed of Na2CO3 salt solution. The system has a selective response to aromatic amino acids, and since the critical concentrations of the three aromatic amino acids that cause ATPS to undergo phase transition are greatly different, the ATPS can be used to detect and identify aromatic amino acids.

[0026] 2. The two-phase aqueous system of the present invention is simple to prepare, environmentally friendly, low-cost, does not require any detection equipment, and has an intuitive effect on the detection and identification of aromatic amino acids and a short time consumption. However, the existing detection technology has an unintuitive detection effect, high requirements on detection equipment, and high cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the phase diagram of S-DPED / SDS / Na2CO3 ATPS. S-DPED :n SDS =2:7(A),n S-DPED :n SDS =4:7(B).

[0028] Figure 2 The two-phase aqueous system (A) and the single-phase system (B) formed by S-DPED / SDS / Na2CO3 before (A) and after (B) the addition of amino acids. The amino acid concentration in Figure B is greater than its critical concentration.

[0029] Figure 3 The distribution of aromatic amino acids below the critical concentration in S-DPED / SDS / Na2CO3 ATPS. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experiments without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0032] S-DPED:(1S,2S)-(-)-1,2-diphenylethylenediamine, CAS:29841-69-8(TCI);

[0033] SDS: sodium dodecyl sulfate, CAS: 151-21-3 (Annaiji);

[0034] Phe: L-phenylalanine, CAS: 63-91-2 (Aladdin);

[0035] Trp: L-tryptophan, CAS: 73-22-3 (Anaiji);

[0036] Tyr: L-tyrosine, CAS: 60-18-4 (TCI).

[0037] Example 1

[0038] Preparation and phase diagram of S-DPED / SDS / Na2CO3 ATPS:

[0039] Using deionized water as solvent, S-DPED, SDS and Na2CO3 were added thereto and completely dissolved at 30°C. A series of (S-DPED+SDS) (2wt.%-10wt.%) / Na2CO3 (8wt.%-22wt.%) solutions were prepared, allowed to stand for equilibrium at 30°C for 10 min, and then centrifuged at 8000 rpm for 5 min to observe whether two phases were formed. Figure 1 The phase diagram of the two-phase aqueous system constructed by S-DPED, SDS and Na2CO3 is shown, wherein the concentration of Na2CO3 is 8wt.% to 22wt.%, Figure 1 n in A S-DPED :n SDS =2:7, Figure 1 B in n S-DPED :n SDS =4:7. When n S-DPED :n SDS When the ratio is 2:7, the concentration of S-DPED is 0.34wt.%-1.74wt.%, and the concentration of SDS is 1.66wt.%-8.26wt.; when n S-DPED :n SDS When the ratio of S-DPED to SDS is 4:7, the concentration of S-DPED is 0.59wt.%-2.96wt.%, and the concentration of SDS is 1.41wt.%-7.04wt.%. When S-DPED / SDS / Na2CO3 forms a stable two-phase aqueous system, the upper phase is mainly composed of S-DPED and SDS solutions, and the lower phase is mainly composed of Na2CO3 solution. In addition, the present invention also observed that n S-DPED :n SDS The formation of S-DPED / SDS / Na2CO3 aqueous two-phase when the ratio is 1:1-1:7. Considering the formation of the aqueous two-phase, the stability of the aqueous two-phase and the volume of the upper and lower phases of the aqueous two-phase, n S-DPED :n SDS =2:7 and n S-DPED :n SDS =4:7 ATPS for subsequent experiments. These two-phase aqueous systems include 0.34wt.%S-DPED / 1.66wt.%SDS / 14.00wt.%Na2CO3ATPS (n S-DPED :nSDS =2:7), 0.70wt.%S-DPED / 3.30wt.%SDS / 14.00wt.%Na2CO3 ATPS(n S-DPED :n SDS =2:7), 0.59wt.%S-DPED / 1.41wt.%SDS / 12.00wt.%Na2CO3 ATPS(n S-DPED :n SDS =4:7), 1.18wt.%S-DPED / 2.82wt.%SDS / 12.00wt.%Na2CO3 ATPS(n S-DPED :n SDS =4:7).

[0040] Example 2

[0041] The critical concentration of aromatic amino acids that causes S-DPED / SDS / Na2CO3 ATPS to transform into a single phase:

[0042] Different concentrations of phenylalanine, tryptophan and tyrosine were added to S-DPED / SDS / Na2CO3 ATPS to observe the effects of these aromatic amino acids on the two-phase aqueous system and record the minimum amino acid concentration (i.e., critical concentration) at which the system transforms from two phases to a single phase. Figure 2 A is S-DPED / SDS / Na2CO3 ATPS without the addition of amino acids, Figure 2 B is a single-phase system formed after adding amino acids to S-DPED / SDS / Na2CO3ATPS and the concentration of the amino acids is greater than its critical concentration.

[0043] The specific experimental steps are as follows:

[0044] (1) Deionized water was used as solvent, S-DPED, SDS and Na2CO3 were added thereto, and completely dissolved at 30°C to prepare 0.34wt.% S-DPED / 1.66wt.% SDS / 14.00wt.% Na2CO3ATPS (n S-DPED :n SDS =2:7) solution, allowed to stand at 30°C for 10 min, then centrifuged at 8000 rpm for 5 min, added different masses of Phe (or Trp or Tyr) to the ATPS, allowed to stand at 30°C for 10 min after complete dissolution, then centrifuged at 8000 rpm for 5 min, wherein the concentration of amino acid is 0-200 μmol / g. Observe the change in the volume of the two phases in the system, and record the critical concentration of amino acid when the system changes from two phases to a single phase.

[0045] Alternatively, deionized water can be used as solvent, and S-DPED, SDS, Na2CO3 and aromatic amino acids can be added thereto and completely dissolved at 30°C to prepare a series of ATPS (n S-DPED :n SDS =2:7), equilibrated at 30°C for 10 min, and then centrifuged at 8000 rpm for 5 min, wherein the concentration of the amino acid is 0-200 μmol / g. The change in the volume of the two phases in the system is observed, and the critical amino acid concentration at which the system changes from two phases to a single phase is recorded.

[0046] (2) Changing the above ATPS to 0.70wt.%S-DPED / 3.30wt.%SDS / 14.00wt.%Na2CO3ATPS(n S-DPED :n SDS =2:7), 0.59wt.%S-DPED / 1.41wt.%SDS / 12.00wt.%Na2CO3 ATPS(n S-DPED :n SDS =4:7), 1.18wt.%S-DPED / 2.82wt.%SDS / 12.00wt.%Na2CO3 ATPS(n S-DPED :n SDS =4:7) Repeat step (1) and record the minimum concentration of amino acids required to transform the system from two phases to a single phase. The results are shown in Table 1. As shown in Table 1, the critical concentrations of aromatic amino acids required to promote the transformation of the above four ATPS into a single phase are quite different, which can be used to detect and identify aromatic amino acids. Among them, these three aromatic amino acids promote 0.70wt.%S-DPED / 3.30wt.%SDS / 14.00wt.%Na2CO3 ATPS(n S-DPED :n SDS =2:7) into a single phase. Therefore, this ATPS has the best detection effect on the three aromatic amino acids.

[0047] Table 1 Critical concentration of amino acids required to induce S-DPED / SDS / Na2CO3 ATPS to transform into a single phase

[0048]

[0049] Example 3

[0050] Distribution of aromatic amino acids in the upper and lower phases of S-DPED / SDS / Na2CO3 ATPS:

[0051] When the amino acid concentration was lower than its critical concentration, their distribution in S-DPED / SDS / Na2CO3ATPS was determined to further understand the selective response of S-DPED / SDS / Na2CO3ATPS to the three aromatic amino acids.

[0052] (1) Determination of Phe, Trp and Tyr standard curves: Prepare 0.70wt.%S-DPED / 3.30wt.%SDS / 14.00wt.%Na2CO3ATPS(n S-DPED :n SDS =2:7) solution to separate the upper and lower phases of ATPS. Then, the lower phase of ATPS is used as a solvent to prepare Phe (or Trp or Tyr) solutions of different concentrations. Phe shows a characteristic absorption peak at 258nm, Trp shows a characteristic absorption peak at 279nm, and Tyr shows a characteristic absorption peak at 293nm. By measuring the absorbance of aromatic amino acids of different concentrations at their characteristic absorption peaks, a standard curve is obtained. Change the above ATPS to 0.34wt.%S-DPED / 1.66wt.%SDS / 14.00wt.%Na2CO3 ATPS(n S-DPED :n SDS =2:7), 0.59wt.%S-DPED / 1.41wt.%SDS / 12.00wt.%Na2CO3 ATPS(n S-DPED :n SDS =4:7), 1.18wt.%S-DPED / 2.82wt.%SDS / 12.00wt.%Na2CO3 ATPS(n S-DPED :n SDS =4:7), repeat the above steps, and the obtained standard curves of Phe, Trp and Tyr were compared with 0.70wt.%S-DPED / 3.30wt.%SDS / 14.00wt.%Na2CO3ATPS(n S-DPED :n SDS =2:7) as the solvent.

[0053] (2) Distribution of amino acids in the two phases of ATPS: When the amino acid concentration was lower than its critical concentration, 0.70 wt.% S-DPED / 3.30 wt.% SDS / 14.00 wt.% Na2CO3 ATPS (n S-DPED :n SDS=2:7) solution, after the two phases are completely separated, the volume of the ATPS lower phase is measured, and the absorbance values ​​corresponding to the characteristic absorption peaks of the three aromatic amino acids in the lower phase are measured by ultraviolet spectrophotometer. The concentration of Phe or Trp or Tyr in the lower phase can be obtained by using the standard curve, and the distribution coefficient of Phe (or Trp or Tyr) can be calculated. The above ATPS is changed to 0.34wt.%S-DPED / 1.66wt.%SDS / 14.00wt.%Na2CO3 ATPS (n S-DPED :n SDS =2:7), 0.59wt.%S-DPED / 1.41wt.%SDS / 12.00wt.%Na2CO3 ATPS(n S-DPED :n SDS =4:7), 1.18wt.%S-DPED / 2.82wt.%SDS / 12.00wt.%Na2CO3 ATPS(n S-DPED :n SDS =4:7), repeat the above steps.

[0054] The calculation formula of the distribution coefficient of Phe, Trp and Tyr is as follows:

[0055] K=(n 总 -C 下 V 下 ) / C 下 V 下 ,

[0056] Where, K: partition coefficient of aromatic amino acids; n 总 : The total molar number of aromatic amino acids in ATPS; C: The concentration of aromatic amino acids in the lower phase of ATPS; V: The volume of the lower phase of ATPS.

[0057] Specifically, deionized water is used as a solvent, and S-DPED, SDS, Na2CO3 and aromatic amino acids are added thereto.

[0058] (2.1) 0.34 wt.% S-DPED / 1.66 wt.% SDS / 14.00 wt.% Na2CO3 ATPS (n) containing 1.68 μmol / g and 3.36 μmol / g of Phe or Trp or Tyr was prepared. S-DPED :n SDS=2:7), wherein the mass concentrations of Phe are 0.28 mg / g and 0.56 mg / g, the mass concentrations of Trp are 0.34 mg / g and 0.69 mg / g, and the mass concentrations of Tyr are 0.30 mg / g and 0.61 mg / g, respectively. After being completely dissolved at 30°C, the mixture is allowed to stand for 10 min and then centrifuged at 8000 rpm for 5 min. The absorbance values ​​of the aromatic amino acids in the lower phase of ATPS at their characteristic absorption peaks are detected, and the concentration of Phe (or Trp or Tyr) in the lower phase is calculated based on the standard curve.

[0059] (2.2) Prepare 0.70 wt.% S-DPED / 3.30 wt.% SDS / 14.00 wt.% Na2CO3 ATPS (n S-DPED :n SDS =2:7), wherein the mass concentrations of Phe are 0.27 mg / g and 0.54 mg / g, the mass concentrations of Trp are 0.33 mg / g and 0.67 mg / g, and the mass concentrations of Tyr are 0.30 mg / g and 0.59 mg / g, and step (2.1) is repeated;

[0060] (2.3) Prepare 0.59wt.% S-DPED / 1.41wt.% SDS / 12.00wt.% Na2CO3ATPS (n S -DPED:n SDS =4:7), wherein the mass concentrations of Phe are 0.28 mg / g and 0.71 mg / g, the mass concentrations of Trp are 0.35 mg / g and 0.88 mg / g, and the mass concentrations of Tyr are 0.31 mg / g and 0.78 mg / g, and step (2.1) is repeated;

[0061] (2.4) Prepare 1.18wt.% S-DPED / 2.82wt.% SDS / 12.00wt.% Na2CO3 ATPS (n S -DPED:n SDS =4:7), wherein the mass concentrations of Phe are 0.28 mg / g and 0.69 mg / g, the mass concentrations of Trp are 0.34 mg / g and 0.86 mg / g, and the mass concentrations of Tyr are 0.30 mg / g and 0.76 mg / g, and step (2.1) is repeated.

[0062] According to the calculation formula of the distribution coefficient, the distribution coefficient of Phe (or Trp or Tyr) in each ATPS is calculated. The results are as follows: Figure 3 As shown. Figure 3 A shows that when n S -DPED:n SDS =2:7, in 0.34wt.%S-DPED / 1.66wt.%SDS / 14.00wt.%Na2CO3 ATPS, when the total concentration of Tyr in the system increased from 1.68μmol / g to 3.36μmol / g, the distribution coefficient of Tyr was almost the same, about 0.05, which indicates that when the concentration of Tyr is lower than its critical concentration, Tyr is almost entirely distributed in the lower phase of ATPS; when the total concentration of Trp in the system increased from 1.68μmol / g to 3.36μmol / g, the distribution coefficient of Trp increased from 0.84 to 1.78, therefore, as the total concentration of Trp in the system increased, the distribution of Trp in the upper phase also increased. When the total concentration of Phe in the system increased from 1.68μmol / g to 3.36μmol / g, the distribution coefficient of Phe remained almost unchanged, about 0.6, therefore, a considerable amount of Phe was in the upper phase of ATPS. Figure 3 B shows that when n S-DPED :n SDS =2:7, in 0.70wt.% S-DPED / 3.30wt.% SDS / 14.00wt.% Na2CO3 ATPS, when the total concentration of Tyr increased from 1.64μmol / g to 3.28μmol / g, the distribution coefficient of Tyr remained almost unchanged at around 0.2, which indicates that when the concentration of Tyr is lower than its critical concentration, Tyr is mainly distributed in the lower phase of ATPS; when the concentration of Trp in the system increased from 1.64μmol / g to 3.28μmol / g, the distribution coefficient of Trp increased from 1.72 to 2.67, therefore, as the concentration of Trp in the system increased, the distribution of Trp in the upper phase increased; when the total concentration of Phe in the system increased from 1.64μmol / g to 3.28μmol / g, the distribution coefficient of Phe remained almost unchanged at around 1, which indicates that the distribution of Phe in the upper and lower phases of ATPS is almost the same. Combining the critical concentrations of the three amino acids in Table 1, it can be seen that among the three aromatic amino acids, Tyr is almost entirely enriched in the lower phase of ATPS, has the least effect on ATPS, and has the highest critical concentration required to promote the transformation of ATPS into a single phase; Trp has the largest distribution in the upper phase of ATPS, and has the most significant effect on ATPS, so its critical concentration for promoting the transformation of ATPS into a single phase is the lowest. Figure 3C and 3D show that the distribution of the three aromatic amino acids in ATPS is less than or equal to 1, and there is no obvious difference in the distribution between the upper and lower phases. Therefore, the critical concentration difference of the three aromatic amino acids in this system is also smaller.

[0063] Combined with Table 1, we can see that when n S-DPED :n SDS =2:7, in 0.70wt.% S-DPED / 3.30wt.% SDS / 14.00wt.% Na2CO3 ATPS, the critical concentration difference of the three aromatic amino acids that causes the two-phase aqueous phase to transform into a single phase is the largest, which is attributed to the obvious difference in their upper and lower phase distribution in the two-phase aqueous phase when the concentration of the three aromatic amino acids is lower than their critical concentration. For aliphatic amino acids (including threonine, alanine, valine, lysine, glutamine, serine, glycine, methionine, aspartic acid, arginine), even if their total concentration in the system is as high as 200μmol / g, they will not cause the system to transform from two phases to a single phase, which further indicates that the two-phase aqueous system is suitable for the detection of the three aromatic amino acids. In subsequent detections, the aromatic amino acids can be identified by observing the minimum concentration of the amino acids required to transform the two-phase aqueous phase into a single phase.

Claims

1. A two-phase aqueous system for detecting aromatic amino acids, characterized in that: The two-phase aqueous system includes S-DPED, SDS and Na2CO3, and the structural formula of the S-DPED is: The structural formula of SDS is:

2. The aqueous two-phase system for detecting aromatic amino acids according to claim 1, characterized in that: The total concentration of S-DPED and SDS in the two-phase aqueous system is 2wt.% to 10wt.%, the concentration of Na2CO3 is 8wt.% to 22wt.%, and deionized water is used as the solvent.

3. The two-phase aqueous system for detecting aromatic amino acids according to claim 1, characterized in that: The concentration of S-DPED in the two-phase aqueous system is 0.34 wt.% to 2.96 wt.%, the concentration of SDS is 1.41 wt.% to 8.26 wt.%, and the concentration of Na2CO3 is 11 wt.% to 19 wt.%.

4. The two-phase aqueous system for detecting aromatic amino acids according to claim 1, characterized in that: The molar ratio of S-DPED to SDS in the aqueous two-phase system is preferably n S-DPED :n SDS =2:7-4:

7.

5. The two-phase aqueous system for detecting aromatic amino acids according to claim 1, characterized in that: The concentration of S-DPED in the two-phase aqueous system is 0.34wt.%, 0.70wt.%, 0.59wt.% or 1.18wt.%, the corresponding concentration of SDS is 1.66wt.%, 3.30wt.%, 1.41wt.% or 2.82wt.%, and the concentration of Na2CO3 is 12.00wt.% or 14.00wt.%.

6. A method for preparing a two-phase aqueous system for detecting aromatic amino acids according to claim 1, characterized in that: Includes steps: Add S-DPED, SDS and Na2CO3 to deionized water, mix well, let stand for equilibrium and then centrifuge to obtain a two-phase aqueous system.

7. Use of the two-phase aqueous system for detecting aromatic amino acids according to claim 1 in detecting and identifying aromatic amino acids.

8. The use according to claim 7, characterized in that: The aromatic amino acid includes any one of phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr).

9. The use according to claim 7, characterized in that: The application is to add aromatic amino acid solutions respectively into a two-phase aqueous system containing S-DPED / SDS / Na2CO3, mix well, balance, and centrifuge.

10. The use according to claim 8, characterized in that: The concentration of the aromatic amino acids is 0-200 μmol / g. The critical concentrations required for three aromatic amino acids, namely phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr), to transform ATPS into a single phase are greatly different. Therefore, the three aromatic amino acids can be identified by the minimum amino acid concentration required for the system to transform from two phases to a single phase. When the amino acid concentration is lower than the critical concentration, tyrosine (Tyr) is mainly distributed in the lower phase of ATPS, tryptophan (Trp) is mainly distributed in the upper phase of ATPS, and the distribution of phenylalanine (Phe) in the upper and lower phases of ATPS is between Tyr and Trp.

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