Tyrosine enantiomer sensor based on aggregation-induced electrochemiluminescence effect of chiral pyrenetetracarboxylic acid and its recognition method

Abstract

The invention discloses a tyrosine enantiomer sensor and an identification method thereof based on the aggregation-induced electrochemiluminescence effect of chiral pyrene tetracarboxylic acid, and belongs to the field of electrochemiluminescence technology. With 3,4,9,10-pyrene tetracarboxylic acid as the main material, S-3-amino-1-butanol as the chiral ligand, chirality transfer is achieved through an amidation reaction, and under the action of dodecyl ammonium bromide, chloroform and cyclohexane, the monomers are aggregated in micelles through intermolecular π-π stacking interactions, which restricts intramolecular motion, thereby triggering the aggregation-induced electrochemiluminescence effect, and obtaining a pyrene tetracarboxylic acid-S-3-amino-1-butanol assembly, potassium persulfate as a co-reaction reagent, when tyrosine enantiomers are present in the system, electrochemiluminescence signals of different intensities are presented, and a sensing method based on a new electrochemiluminescence system of a pyrene tetracarboxylic acid-S-3-amino-1-butanol assembly is constructed accordingly, and stable identification of tyrosine enantiomers is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemiluminescence, and in particular relates to a tyrosine enantiomer recognition method based on the electrochemiluminescence effect induced by aggregation of chiral materials of pyrenetetracarboxylic acid. Background Art

[0002] Chirality is one of the key features of nature and plays an important role at the molecular level in biology, medicine, and biotechnology. Amino acids (AAs) are the most important and predominant chiral compounds in nature and can serve as important biomarkers for various metabolic diseases. Tyrosine (L-Tyr) is an important ketogenic glycogen amino acid and an important precursor for various bioactive molecules such as epinephrine, norepinephrine, and dopamine, all of which play a role in regulating mood. Importantly, the accumulation or metabolism of L-Tyr in the body is associated with a variety of diseases that may disrupt cellular homeostasis and contribute to the pathogenesis of diseases such as Huntington's disease, gastroesophageal malignancies, and liver cancer. Accurate monitoring of tyrosine concentration is very important for health assessment. Therefore, the chiral recognition of amino acid enantiomers is of great significance in life sciences. Currently, a variety of analytical techniques such as chromatography, nuclear magnetic resonance, fluorescence spectroscopy, capillary electrophoresis, and electrochemistry have been applied to the chiral recognition of amino acid enantiomers. Electrochemical chiral sensing has the advantages of low cost, fast response, high sensitivity, simple operation, and easy regeneration. There are few studies on chiral recognition sensing based on ECL, mainly because there is a lack of chiral recognizers with both high enantiomeric recognition ability and good electrochemical response. Therefore, this paper synthesized a new ECL luminophore, a structurally stable pyrenetetracarboxylic acid-S-3-amino-1-butanol assembly, for tyrosine enantiomer recognition. Summary of the invention

[0003] The present invention aims to provide a tyrosine enantiomer recognition method based on the aggregation-induced electrochemiluminescence effect of chiral pyrenetetracarboxylic acid. 3,4,9,10-pyrenetetracarboxylic acid is used as a main material, S-3-amino-1-butanol is used as a chiral ligand, chirality transfer is achieved through an amidation reaction, and under the action of dodecyltrimethylammonium bromide, chloroform and cyclohexane, monomers are aggregated in micelles through intermolecular π-π stacking interactions, and intramolecular motion is restricted, thereby triggering the aggregation-induced electrochemiluminescence effect to obtain a pyrenetetracarboxylic acid-S-3-amino-1-butanol assembly. Potassium persulfate is used as a co-reaction reagent. When tyrosine enantiomers exist in the system, electrochemiluminescence signals of different intensities are presented. Based on this, a sensing method based on a new electrochemiluminescence system of the pyrenetetracarboxylic acid-S-3-amino-1-butanol assembly is constructed, and the method has the advantages of being simple, sensitive, selective and highly stable for tyrosine enantiomers.

[0004] The present invention provides the following technical solutions:

[0005] A method for identifying tyrosine enantiomers based on the aggregation-induced electrochemiluminescence effect of chiral pyrenetetracarboxylic acid comprises the following steps:

[0006] S1: Add perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) to a NaOH solution, stir in a water bath until the solution turns yellow-green, add HCl until complete precipitation, centrifuge with ultrapure water, wash, and dry the product to obtain 3,4,9,10-pyrenetetracarboxylic acid;

[0007] S2: dissolving 3,4,9,10-pyrenetetracarboxylic acid (PTCA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), and N-hydroxysuccinimide (NHS) in N,N-dimethylformamide (DMF) and stirring;

[0008] S3: mixing S-3-amino-1-butanol (S-3AB) with the above solution under stirring;

[0009] S4: The solid obtained by centrifugation is washed and dried to obtain pyrenetetracarboxylic acid-S-3-amino-1-butanol (PTCA-S-3AB);

[0010] S5: Mix pyrenetetracarboxylic acid-S-3-amino-1-butanol, chloroform, cyclohexane, and dodecyltrimethylammonium bromide (DTAB) under stirring conditions, stir at 60-90°C, react for 1-3 hours, centrifuge the obtained solid and wash it to obtain pyrenetetracarboxylic acid-S-3-amino-1-butanol assembly (AG-PTCA-S-3AB).

[0011] Further, in step S1, the concentration of NaOH is 0.5-2.0M, and the concentration of HCl is 0.5-2.0M; preferably, the concentration of NaOH is 0.5M; and the concentration of HCl is 1M;

[0012] Furthermore, in step S2, the molar ratio of 3,4,9,10-pyrenetetracarboxylic acid to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(5-8), and the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:(2-5); preferably, the molar ratio of perylene-3,4,9,10-tetracarboxylic dianhydride (PTCA) to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) is 3:18:45.

[0013] Further, S-3-amino-1-butanol is dissolved in N,N-dimethylformamide, stirred at 20-50° C., and reacted for 6-15 hours; the molar ratio of PTCA to S-3-amino-1-butanol (S-3AB) is 1:3-4;

[0014] Preferably, in step S3, the concentration of S-3AB is 12 mM, and the stirring time after adding EDC·HCl and NHS is 15 min.

[0015] Furthermore, the drying time in step S4 is 4-8 hours. Preferably, in step S4, the centrifugal speed is 12000 rpm and the drying time is 6 hours.

[0016] Further, in step S5, stirring is performed at 60-90°C for 1-3h. Preferably, in step S5, the reaction time is 1h and the reaction temperature is 70°C.

[0017] The application for the identification of tyrosine enantiomers is as follows:

[0018] The pyrenetetracarboxylic acid-S-3-amino-1-butanol assembly is dispersed in deionized water, mixed with a certain volume of tetra-n-octylammonium bromide (TOAB), and the mixed solution is dropped on the surface of a glassy carbon electrode and dried at room temperature to obtain AG-PTCA-S-3AB / GCE; the concentration of the mixed solution is 0.1-2 mg / mL and the mass is 0.5-10 μg.

[0019] A three-electrode system was composed of AG-PTCA-S-3AB / GCE as the working electrode, platinum wire as the counter electrode, and Ag / AgCl electrode as the reference electrode. AG-PTCA-S-3AB / GCE was used as the working electrode, the Ag / AgCl electrode was used as the reference electrode, the platinum sheet electrode was used as the counter electrode, phosphate buffered saline (PBS) was used as the electrolyte, and potassium persulfate was used as the co-reactant. The electrochemiluminescence intensity of AG-PTCA-S-3AB / GCE in PBS electrolyte containing L / D-tyrosine was measured by an electrochemiluminescence instrument.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The tyrosine enantiomer recognition method based on the aggregation-induced electrochemiluminescence effect of chiral pyrenetetracarboxylic acid provided by the present invention realizes the aggregation-induced electrochemiluminescence effect of chiral materials through surfactant-induced self-assembly, and has better electrochemiluminescence performance and better recognition effect;

[0022] (2) The method for identifying tyrosine enantiomers based on the electrochemiluminescence effect of chiral materials induced by aggregation of pyrenetetracarboxylic acid provided by the present invention uses a glassy carbon electrode modified with a pyrenetetracarboxylic acid-S-3-amino-1-butanol assembly as a working electrode, which can achieve selective identification of tyrosine enantiomers, with reliable results, good reproducibility, high stability, high sensitivity and simple operation method.

[0023] (3) The tyrosine enantiomer recognition method based on the electrochemiluminescence effect of the chiral material aggregation induced by pyrenetetracarboxylic acid provided by the present invention can achieve specific recognition of tyrosine. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The scanning electron microscope images of PTCA, PTCA-S-3AB and AG-PTCA-S-3AB in Example 1;

[0025] Figure 2 The infrared spectra of PTCA, PTCA-S-3AB and AG-PTCA-S-3AB in Example 1;

[0026] Figure 3 This is a comparison chart of ECL performance of different materials in Example 1;

[0027] Figure 4 This is the ECL diagram of PTCA-S-3AB recognizing tyrosine enantiomers in Comparative Example 1;

[0028] Figure 5 This is the ECL diagram of AG-PTCA-S-3AB recognizing tyrosine enantiomers in Example 1;

[0029] Figure 6 This is a bar graph showing the recognition of different amino acids by the AG-PTCA-S-3AB pair in Example 1. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0031] Example

[0032] 0.1 g of perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA, molecular weight 392.32) was added to 10 mL of 0.5 M NaOH solution, stirred in a water bath at 80 ° C for 1 hour until the solution turned yellow-green, and then 1.0 M HCl was added dropwise to the mixed solution until complete precipitation, the pH was kept slightly acidic, and washed by centrifugation with ultrapure water to remove excess reagents, and dried at 60 ° C. The prepared product is PTCA. 12.8 mg of 3,4,9,10-pyrenetetracarboxylic acid (PTCA, molecular weight 428.34), 34 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl, molecular weight 191.7) and 51 mg of N-hydroxysuccinimide (NHS, molecular weight 115.09) were weighed and dissolved in 10 mL of DMF and stirred for 15 min to activate the carboxyl group on PTCA. 10 mg of S-3-amino-1-butanol (S-3AB, molecular weight 89.13) was added and stirred overnight to allow amidation reaction to occur. The mixture was heated to 40 ℃ and 10 ℃ for 2 hours. The mixture was centrifuged repeatedly, washed, and dried at 60°C to obtain PTCA-S-3AB. 0.5 mg PTCA-S-3AB was ultrasonically dispersed in 2 mL ultrapure water. 20 mg dodecyltrimethylammonium bromide (DTAB), 800 μL chloroform and 200 μL cyclohexane were added and stirred at room temperature for 30 s. 2 mL ultrapure water was added and the organic phase was evaporated for 1 h under stirring at 70°C in a water bath. After cooling to room temperature, the mixture was washed by centrifugation with ultrapure water to obtain a solid, which was the PTCA-S-3AB assembly (AG-PTCA-S-3AB). The solid was redispersed in ultrapure water.

[0033] 0.5 mg AG-PTCA-S-3AB was dispersed in 0.5 mL ultrapure water, ultrasonically dispersed for 5 min, an equal volume of 10 mM tetraoctylammonium bromide (TOAB) was added and mixed evenly, 8 μL of the dispersed mixed solution was drop-coated on the surface of GCE, and dried under an infrared lamp. The final prepared modified electrode was marked as AG-PTCA-S-3AB / GCE. With AG-PTCA-S-3AB / GCE as the working electrode, Ag / AgCl electrode as the reference electrode, platinum electrode as the counter electrode, PBS with a pH of 7 as the electrolyte, and 18 mM potassium persulfate as the co-reaction reagent, the intensity of electrochemiluminescence (ECL) of AG-PTCA-S-3AB / GCE was tested by an electrochemiluminescence instrument.

[0034] AG-PTCA-R-3AB was synthesized by the same method by replacing S-3-amino-1-butanol with (R)-3-amino-1-butanol. The final modified electrode was labeled AG-PTCA-S-3AB / GCE, which did not have a good recognition effect on tyrosine enantiomers.

[0035] Comparative Example

[0036] 12.8 mg of 3,4,9,10-pyrenetetracarboxylic acid (PTCA, molecular weight 428.34), 34 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, molecular weight 191.7) and 51 mg of N-hydroxysuccinimide (NHS, molecular weight 115.09) were weighed and dissolved in 10 mL of DMF, and stirred for 15 min to activate the carboxyl group on PTCA, and 10 mg of S-3-amino-1-butanol (S-3AB, molecular weight 89.13) was added, and stirring was continued overnight. The carboxyl group on PTCA reacted with the amino group on S-3AB for amidation reaction, and the mixture was repeatedly centrifuged with ethanol and ultrapure water, washed, and dried at 60°C to obtain PTCA-S-3AB.

[0037] 0.5 mg PTCA-S-3AB was dispersed in 0.5 mL ultrapure water, ultrasonically dispersed for 5 min, an equal volume of TOAB was added and mixed evenly, 8 μL of the dispersed mixed solution was drop-coated on the GCE surface, and dried under an infrared lamp. The final modified electrode was labeled as PTCA-S-3AB / GCE. With PTCA-S-3AB / GCE as the working electrode, Ag / AgCl electrode as the reference electrode, platinum electrode as the counter electrode, PBS with a pH of 7 as the electrolyte, and 18 mM potassium persulfate as the co-reactant, the intensity of electrochemiluminescence (ECL) of PTCA-S-3AB / GCE was tested by an electrochemiluminescence instrument.

[0038] Experimental data analysis

[0039] like Figure 1 , where A is the SEM image of PTCA; B and C are SEM images of PTCA-S-3AB at different scales; D and E are SEM images of AG-PTCA-S-3AB at different scales. As can be seen from Figure A, PTCA presents an irregular block structure, the morphology of PTCA-S-3AB presents a regular rod-like structure with uniform distribution, and the morphology of AG-PTCA-S-3AB becomes smoother than that of PTCA-S-3AB. As can be seen from Figures C and E, the monomer PTCA-S-3AB aggregates to form AG-PTCA-S-3AB, which has better ECL performance.

[0040] like Figure 2 The infrared spectra of different samples are shown in Figure 1. PTCA is at 3440 cm -1 The absorption peak that appears is the stretching vibration peak of -OH, at 1770cm -1 The absorption peak at 1080 cm is the stretching vibration of C=O. -1 The absorption peak at 1639cm is the characteristic absorption peak of CN.-1 The bending vibration peak of NH is 3446cm -1 is the stretching vibration peak of the NH bond in the amide bond, indicating that an amide bond is formed between PTCA and S-3AB. The peaks of AG-PTCA-3AB are 1076 cm -1 , 1647cm -1 , 1772cm -1 , 3446cm -1 An absorption peak appeared, which was slightly shifted compared to PTCA-S-3AB. This was because during the assembly process, the material aggregated, causing a slight shift in the infrared absorption peak.

[0041] like Figure 3 This is a comparison chart of the ECL performance of different materials. It can be seen that the bare electrode and PTCA have no obvious ECL behavior. When PTCA-S-3AB is formed after amidation, the ECL performance is slightly improved. The performance of AG-PTCA-3AB after assembly is significantly enhanced. This is due to aggregation-induced emission, which leads to a significant change in the ECL signal. It can be seen that the final composite material has excellent luminescence performance.

[0042] like Figure 4 This is the ECL comparison chart of PTCA-3AB in the comparative example in the PBS solution containing tyrosine enantiomers. It can be found that when D-Tyr exists in the solution, the ECL signal decreases from 593 a.u. to 393 a.u., and when L-Tyr exists in the solution, the ECL signal decreases to 309 a.u., indicating that PTCA-S-3AB prefers to bind to L-Tyr.

[0043] like Figure 5 This is the ECL comparison chart of AG-PTCA-3AB in Example 1 in a PBS solution containing tyrosine enantiomers. It can be seen that when D-Tyr is present in the solution, the ECL intensity decreases slightly, and the ECL decreases from 3247 a.u. to 2322 a.u., while when L-Tyr is present in the solution, the ECL intensity decreases significantly, and the ECL signal decreases to 1231 a.u. This is because the material is more inclined to bind to L-Tyr, resulting in the enrichment of L-Tyr on the electrode surface. Due to the poor electron transport ability of amino acids, the ECL signal is reduced.

[0044] like Figure 6 The recognition ability of AG-PTCA-3AB for different amino acids in Example 1 is investigated. It can be seen that the ECL of tryptophan, phenylalanine and proline D / ECL L It is close to 1, indicating that the material AG-PTCA-3AB has almost no recognition ability for these three amino acid enantiomers, while the ECL for tyrosine enantiomer isD / ECL L The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and deformations can be made without departing from the technical principles of the present invention, and these improvements and deformations should also be regarded as the protection scope of the present invention.

Claims

1. A sensor for identifying tyrosine enantiomers by electrochemiluminescence effect, characterized in that: First, pyrenetetracarboxylic acid-S-3-amino-1-butanol is prepared, and pyrenetetracarboxylic acid-S-3-amino-1-butanol monomers are aggregated in micelles to obtain pyrenetetracarboxylic acid-S-3-amino-1-butanol assemblies; The pyrenetetracarboxylic acid-S-3-amino-1-butanol assembly was dispersed in deionized water and mixed with tetra-n-octylammonium bromide. The mixture was drop-coated on the surface of a glassy carbon electrode and dried to obtain AG-PTCA-S-3AB / GCE, which was used as a working electrode to identify tyrosine enantiomers based on the aggregation-induced electrochemiluminescence method of chiral pyrenetetracarboxylic acid. The specific preparation method of the pyrenetetracarboxylic acid-S-3-amino-1-butanol assembly is: a. Add perylene-3,4,9,10-tetracarboxylic dianhydride to a NaOH solution and stir in a water bath until the solution turns yellow-green, add HCl solution until it is completely precipitated, centrifuge with ultrapure water, wash, and dry the product to obtain 3,4,9,10-pyrenetetracarboxylic acid; b. dissolving 3,4,9,10-pyrenetetracarboxylic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide in N,N-dimethylformamide; c. mixing the S-3-amino-1-butanol solution with the solution in step b under stirring; d. After the reaction in step c, centrifugation is performed, and the resulting solid is washed and dried to obtain pyrenetetracarboxylic acid-S-3-amino-1-butanol; e. Under stirring conditions, pyrenetetracarboxylic acid-S-3-amino-1-butanol, chloroform, cyclohexane, and dodecyltrimethylammonium bromide were stirred for reaction, and the solid obtained by centrifugation was washed to obtain a pyrenetetracarboxylic acid-S-3-amino-1-butanol assembly.

2. The sensor for identifying tyrosine enantiomers by electrochemiluminescence effect according to claim 1, characterized in that: The concentration of the NaOH solution in step a is 0.5-2.0 M, and the concentration of the HCl solution is 0.5-2.0 M.

3. The sensor for identifying tyrosine enantiomers by electrochemiluminescence effect according to claim 1, characterized in that: In step b, the molar ratio of 3,4,9,10-pyrenetetracarboxylic acid to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(5-8), and the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:(2-5).

4. The sensor for identifying tyrosine enantiomers by electrochemiluminescence effect according to claim 1, characterized in that: In step c, the S-3-amino-1-butanol is dissolved in step b, and the mixture is stirred and reacted at 20-50° C. for 6-15 hours; the molar ratio of PTCA to S-3-amino-1-butanol (S-3AB) is 1:3-4.

5. The sensor for identifying tyrosine enantiomers by electrochemiluminescence effect according to claim 1, characterized in that: The stirring reaction conditions in step e are: stirring the reaction at 60-90° C. for 1-3 h.

6. The method for identifying tyrosine enantiomers by electrochemiluminescence effect of a sensor according to any one of claims 1 to 5, characterized in that: Using AG-PTCA-S-3AB / GCE as the working electrode, Ag / AgCl electrode as the reference electrode, platinum electrode as the counter electrode, phosphate buffered saline as the electrolyte, and potassium persulfate as the co-reactant, the electrochemiluminescence intensity of the pyrenetetracarboxylic acid-S-3-amino-1-butanol assembly in phosphate electrolyte containing L / D-tyrosine was measured by an electrochemiluminescence instrument for the identification of tyrosine enantiomers.

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