Method for detecting tryptophan enantiomers using a chiral electrochemical sensor constructed by ion pair interaction

Abstract

The present application belongs to the field of electrochemical detection, and particularly relates to a method for detecting tryptophan enantiomers by a chiral electrochemical sensor constructed by ion pair interaction. The present application provides a method for detecting tryptophan enantiomers by a chiral electrochemical sensor constructed by ion pair interaction, which utilizes DNA as a chiral selector, and realizes the recognition and detection of tryptophan enantiomers based on the different binding abilities of ion pairs formed by two tryptophan enantiomers and amine compounds to DNA, and further causes the difference of peak current signals of the two tryptophan enantiomers in voltammetry to realize the recognition and detection of tryptophan enantiomers. The sensor has the advantages of simple construction, high sensitivity and low cost, and provides a new idea and method for the recognition and detection of tryptophan enantiomers in actual samples. The present application provides a new way for constructing an electrochemical chiral sensor, and shows that the sensor has great potential in the application of chiral recognition.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical detection, specifically relating to a method for detecting tryptophan enantiomers using a chiral electrochemical sensor constructed from ion-pair interactions. Background Technology

[0002] Chirality is a fundamental phenomenon common in nature and a property possessed by most biomolecules. Due to their different spatial configurations, chiral molecules exhibit different effects on organisms; one enantiomer is beneficial, while the other is useless or even harmful. Therefore, developing an effective and rapid method for identifying enantiomers is crucial and has significant implications for advancements in food, environment, and medicine.

[0003] As is well known, amino acids are the basic structural units of proteins and essential nutrients for the growth of organisms. Except for glycine, all naturally occurring amino acids are chiral molecules. For example, tryptophan is an essential amino acid; however, only L-amino acids can be directly utilized by organisms, and they are involved in metabolism, immune responses, and neuroendocrine processes. D-amino acids are non-protein amino acids that are difficult for organisms to utilize, do not participate in metabolism, and may even have adverse effects on living systems. However, D-amino acids can serve as important precursors for anticancer and immunomodulatory drugs and are mainly found in microorganisms and green plants. Therefore, the identification of tryptophan enantiomers has received widespread attention. To date, various methods have been developed to identify tryptophan enantiomers, such as chromatography, spectroscopy, capillary electrophoresis, ultraviolet spectrophotometry, and electrochemical methods.

[0004] One of the main goals of electrochemical chiral recognition is to construct an efficient chiral selector that has recognition sites for certain chiral enantiomers. Currently, various materials are available for chiral selectors of amino acid chiral recognition, such as bovine serum albumin, cyclodextrin, molecularly imprinted polymers, and crown ethers. Deoxyribonucleic acid (DNA), as a natural chiral macromolecule, has a double helix structure, typically in a right-handed helical conformation. DNA has been found to exhibit specific chiral recognition of certain chiral molecules, including antibiotics, amino acids, and metal supramolecular complexes. However, relying solely on DNA for chiral molecule recognition is relatively inefficient. Summary of the Invention

[0005] To address the problem of low recognition efficiency in existing methods for identifying chiral molecules, this invention provides a method for detecting tryptophan enantiomers using a chiral electrochemical sensor constructed from ion-pair interactions.

[0006] The method for detecting tryptophan enantiomers using a chiral electrochemical sensor constructed from ion-pair interactions utilizes DNA as a chiral selector. Based on the fact that ion pairs formed by D- / L-Trp (tryptophan enantiomers) and amine compounds have different binding abilities to DNA, the peak current signals of the two tryptophan enantiomers in the voltammetry diagram show differences, thereby enabling the identification and detection of tryptophan enantiomers.

[0007] In the method for detecting tryptophan enantiomers using a chiral electrochemical sensor constructed from the above ion-pair interactions, the mass percentage of D-Trp (D-tryptophan) and L-Trp (L-tryptophan) in the D- / L-Trp is 0% to 100%.

[0008] The method for detecting tryptophan enantiomers using a chiral electrochemical sensor constructed from ion-pair interactions comprises the following steps: inserting the electrode dsDNA / AuNPs / GCE into a PBS buffer containing D- / L-Trp and amine compounds; performing electrochemical detection using differential pulse voltammetry (DPV); a scan potential of 0.4–1.1 V; and a pulse amplitude of 50 mVs. –1 The pulse width is 0.05s, and the peak current ratio (I) is used. L-Trp / I D-Trp To evaluate recognition efficiency.

[0009] In the above operation steps, the amine compound in the PBS buffer containing D- / L-Trp and the amine compound is any one of ethylamine, diethylamine, or triethylamine; preferably, the amine compound is TEA (triethylamine). The equivalent ratio of D- / L-Trp to the amine compound is 1:0.1 to 1:2.5; preferably, the equivalent ratio of D- / L-Trp to the amine compound is 1:1.5. Most preferably, the equivalent ratio of D- / L-Trp to triethylamine is 1:1.5. The PBS buffer is a mixed solution of 10mM Na2HPO4, 10mM NaH2PO4, and 0.1M KCl, with a pH of 7.0.

[0010] In the above operation steps, the preparation method of the electrode dsDNA / AuNPs / GCE is as follows:

[0011] a. The GCE (glassy carbon electrode) was first polished on chamois cloth with Al2O3 powder of 0.3 μm and 0.05 μm in sequence, and then ultrasonically cleaned in acetone, anhydrous ethanol and ultrapure water in sequence, and dried with N2. Gold nanoparticles were deposited on the surface of the glassy carbon electrode using the time-current curve method (Amperometric i-t curve) to obtain the electrode AuNPs / GCE.

[0012] b. At room temperature, drop 10 μL of PBS buffer containing 1 μM ssDNA (single-stranded DNA) onto the AuNPs / GCE electrode and protect it from light for 12 hours. Then rinse the electrode with PBS buffer and ultrapure water to remove ssDNA that has not bound to the electrode, and dry it under N2 flow to obtain the electrode ssDNA / AuNPs / GCE.

[0013] c. Drop 10 μL Tris-HCl buffer containing 0.9 mM MCH (6-mercapto-1-hexanol) onto the surface of the electrode ssDNA / AuNPs / GCE, and incubate in the dark for 60 minutes to obtain the electrode MCH / ssDNA / AuNPs / GCE.

[0014] d. Dissolve 1 μM of ssDNA complementary to the ssDNA described in step c in 10 μL of SSC buffer solution, and then drop it onto the surface of electrode MCH / ssDNA / AuNPs / GCE. Incubate at 37°C in the dark for 1 h to obtain electrode dsDNA / AuNPs / GCE.

[0015] In the above-mentioned preparation method of electrode dsDNA / AuNPs / GCE, step a describes the deposition of gold nanoparticles on the surface of the glassy carbon electrode. The operation steps are as follows: insert the platinum wire electrode, Ag / AgCl electrode and glassy carbon electrode into a 2mM chloroauric acid solution in the dark, apply a constant voltage of -0.2V for 90 seconds, rinse off the excess solution on the electrode surface, clean the electrode with ultrapure water, and dry it with N2.

[0016] In the above method for preparing the electrode dsDNA / AuNPs / GCE, the PBS buffer in step b is a mixed solution of 10mM Na2HPO4, 10mM NaH2PO4 and 0.1M KCl, with a pH of 7.0. The ssDNA is dissolved in the PBS buffer and stored at -20℃ for later use.

[0017] In the above method for preparing electrode dsDNA / AuNPs / GCE, the ssDNA base sequence in step b is as follows:

[0018] P1:5'-SH-(CH2)6-GGGGCCGGGG-3', with an SH-(CH2)6-substituent group on the G at the 5' end;

[0019] P2:5'-CCCCGGCCCC-3';

[0020] P3:5'-SH-(CH2)6-GCGCGCGCGC-3', with an SH-(CH2)6-substituent group on the G at the 5' end;

[0021] P4:5'-GCGCGCGCGC-3';

[0022] P5:5'-SH-(CH2)6-AAAATTAAAA-3', with an SH-(CH2)6-substituent group on the G at the 5' end;

[0023] P6:5'-TTTTAATTTT-3';

[0024] P7:5'-SH-(CH2)6-ATATATATAT-3', with an SH-(CH2)6-substituent group on the G at the 5' end;

[0025] P8:5'-ATATATATAT-3';

[0026] Among them, ssDNA-P1 and ssDNA-P2 are complementary to obtain dsDNA-P1&P2; that is, when the ssDNA in step b is ssDNA-P1, the ssDNA that is complementary to the ssDNA in step d and step c is ssDNA-P2.

[0027] ssDNA-P3 and ssDNA-P4 are complementary to obtain dsDNA-P3&P4; that is, when the ssDNA in step b is ssDNA-P3, the ssDNA that is complementary to the ssDNA in step d and step c is ssDNA-P4.

[0028] ssDNA-P5 and ssDNA-P6 are complementary to obtain dsDNA-P5&P6; that is, when the ssDNA in step b is ssDNA-P5, the ssDNA that is complementary to the ssDNA in step d and step c is ssDNA-P6.

[0029] ssDNA-P7 and ssDNA-P8 are complementary to obtain dsDNA-P7&P8; that is, when the ssDNA in step b is ssDNA-P7, the ssDNA that is complementary to the ssDNA in step d and step c is ssDNA-P8.

[0030] In the above method for preparing electrode dsDNA / AuNPs / GCE, the Tris-HCl buffer in step c is 10 mM with a pH of 7.0.

[0031] In the above method for preparing the electrode dsDNA / AuNPs / GCE, the SSC buffer solution in step d is a mixed solution of 0.15M sodium chloride and 15mM sodium citrate, with a pH of 7.0. The ssDNA complementary to the ssDNA described in step c is dissolved in the SSC buffer and stored at -20°C for later use.

[0032] This invention provides a novel chiral electrochemical sensor for the identification and detection of tryptophan using ion-pair interactions. By selecting appropriate compounds to form ion pairs with tryptophan enantiomers, the spatial stereostructure of the tryptophan enantiomers is altered, cleverly enhancing the stereoselective binding ability of dsDNA at the sensing interface to D- / L-Trp (tryptophan enantiomers). This results in a sensor capable of both chiral recognition and content determination. Furthermore, the sensor exhibits good stability and high selectivity. This sensor is simple to construct, sensitive, and inexpensive, providing a new approach and method for the identification and detection of tryptophan enantiomers in practical samples. This invention offers a new pathway for constructing electrochemical chiral sensors, demonstrating their significant potential in chiral recognition applications. Attached Figure Description

[0033] Figure 1 A schematic diagram of the construction of dsDNA / AuNPs / GCE for recognizing tryptophan enantiomers.

[0034] Figure 2 The effect of electrodes modified with different dsDNA sequences on tryptophan enantiomer recognition: a) electrode dsDNA-P1&P2 / AuNPs / GCE; b) electrode dsDNA-P3&P4 / AuNPs / GCE; c) electrode dsDNA-P5&P6 / AuNPs / GCE; d) dsDNA-P7&P8 / AuNPs / GCE.

[0035] Figure 3 The effect of different amines on the recognition of tryptophan enantiomers: a) ethylamine; b) diethylamine; c) triethylamine.

[0036] Figure 4 The effect of different concentrations of DNA on tryptophan enantiomer recognition.

[0037] Figure 5 The effect of different pH values ​​on the recognition of tryptophan enantiomers.

[0038] Figure 6 The effect of ion pair binding time on tryptophan enantiomer recognition.

[0039] Figure 7 The effect of different proportions of triethylamine on the recognition of tryptophan enantiomers.

[0040] Figure 8 A: Linear relationship between peak current and D-Trp% Graph. B: DPV graph based on electrode dsDNA / AuNPs / GCE with different D-Trp% values.

[0041] Figure 9A: Linear correlation graph of peak current with L-Trp and D-Trp concentrations. B: DPV graph based on electrode dsDNA / AuNPs / GCE in L-Trp concentrations. C: DPV graph based on electrode dsDNA / AuNPs / GCE in D-Trp concentrations.

[0042] Figure 10 Stability study based on electrode dsDNA-P1&P2 / AuNPs / GCE.

[0043] Figure 11 Anti-interference study based on electrode dsDNA-P1&P2 / AuNPs / GCE.

[0044] Figure 12 The effect of adding different amino acids on optical rotation. Detailed Implementation

[0045] A novel chiral electrochemical sensor constructed using ion-pair interactions is proposed for the detection of tryptophan. The specific operational steps are as follows: The electrode dsDNA / AuNPs / GCE is inserted into a PBS buffer containing tryptophan and amine compounds. Electrochemical detection is performed using differential pulse voltammetry (DPV), with a scan potential of 0.4–1.1 V and a pulse amplitude of 50 mVs. –1 The pulse width is 0.05s, and the peak current ratio (I) is used. L-Trp / I D-Trp To evaluate recognition efficiency.

[0046] In the above operation steps, the amine compound in the PBS buffer containing D- / L-Trp and the amine compound is any one of ethylamine, diethylamine, or triethylamine; preferably, the amine compound is TEA (triethylamine). The equivalent ratio of D- / L-Trp to the amine compound is 1:0.1 to 1:2.5; preferably, the equivalent ratio of D- / L-Trp to the amine compound is 1:1.5. Most preferably, the equivalent ratio of D- / L-Trp to triethylamine is 1:1.5. The PBS buffer is a mixed solution of 10mM Na2HPO4, 10mM NaH2PO4, and 0.1M KCl, with a pH of 7.0.

[0047] L-Tryptophan (L-Trp, 99%), D-Tryptophan (D-Trp, 98%), L-Phenylalanine (L-Phe, 99%), L-Lysine (L-Lys, 98%), L-Aspartic acid (L-Asp, 99%), and glycine (Gly, 99.5%) were all purchased from Shanghai Maclean's Biochemical Technology Co., Ltd. Chloroauric acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. All water used in the experiments was ultrapure water. Tryptophan solution was prepared with 10mM PBS buffer. ssDNA was obtained from Shanghai Sangon Biotech Co., Ltd.

[0048] A conventional three-electrode system was used, with either a bare gold electrode and a modified gold electrode (AuE, 3 mm) or a bare glassy carbon electrode and a modified glassy carbon electrode (GCE, 3 mm) as the working electrode, for differential pulse voltammetry (DPV) electrochemical measurements. A platinum wire was used as the counter electrode, and Ag / AgCl was used as the reference electrode. The absorbance of tryptophan was measured using a UV-2600 ultraviolet spectrophotometer. The optical rotation of mixed amino acids was measured using an Anton Paar MCP 100 fully automated polarimeter.

[0049] Example 1: Preparation of electrode dsDNA-P1&P2 / AuNPs / GCE

[0050] The glassy carbon electrode was first polished on chamois cloth with Al2O3 powder of 0.3 μm and 0.05 μm in sequence, and then ultrasonically cleaned in acetone, anhydrous ethanol and ultrapure water in sequence, and dried with N2 for later use.

[0051] Gold nanoparticles were deposited on the surface of a glassy carbon electrode using the time-current curve method (Amperometric it curve). A platinum wire electrode, an Ag / AgCl electrode, and a glassy carbon electrode were immersed in a 2 mM chloroauric acid solution (protected from light). A constant voltage of -0.2 V was applied for 90 seconds, excess solution was rinsed off the electrode surface, the electrodes were cleaned with ultrapure water, and dried with N2. The resulting electrode was designated "AuNPs / GCE".

[0052] At room temperature, 10 μL of PBS buffer containing 1 μM ssDNA-P1 was dropped onto the electrode "AuNPs / GCE" and incubated in the dark for 12 hours to allow DNA to self-assemble onto the gold electrode surface using S-Au. The electrode was then rinsed with PBS buffer and ultrapure water to remove unbound ssDNA-P1 and dried under N2 flow. The modified electrode was named "ssDNA-P1 / AuNPs / GCE". Next, 10 μL of Tris-HCl buffer containing 0.9 mM MCH was added to the electrode surface and incubated in the dark for 60 minutes to block the active site of the electrode. The resulting electrode was designated "MCH / ssDNA-P1 / AuNPs / GCE". Finally, 10 μL of LSSC buffer (pH 7.0) containing 1 μM ssDNA-P2 was dropped onto the electrode surface and incubated at 37°C in the dark for 1 hour to allow DNA hybridization, resulting in the electrode "dsDNA-P1&P2 / AuNPs / GCE".

[0053] The preparation methods for electrodes dsDNA-P3&P4 / AuNPs / GCE, dsDNA-P5&P6 / AuNPs / GCE, and dsDNA-P7&P8 / AuNPs / GCE are the same as those for electrodes dsDNA-P1&P2 / AuNPs / GCE.

[0054] Triethylamine was added dropwise to the PBS buffer containing D- / L-Trp (the equivalent ratio of triethylamine to D- / L-Trp was 1:1.5), and the mixture was shaken continuously for 10 min to obtain a PBS buffer containing D- / L-Trp and the amine compound triethylamine.

[0055] The modified electrode “dsDNA-P1&P2 / AuNPs / GCE” was inserted into PBS buffer containing D- / L-Trp and triethylamine. Electrochemical detection was performed using differential pulse voltammetry (DPV), with a scan potential of 0.4–1.1 V and a pulse amplitude of 50 mVs. –1 The pulse width is 0.05s, and the peak current ratio (I) is used. L-Trp / I D-Trp To evaluate recognition efficiency.

[0056] Example 2: Effect of dsDNA sequence on tryptophan enantiomer recognition efficiency

[0057] To investigate whether different dsDNA sequences affect the recognition efficiency of tryptophan enantiomers, four different dsDNA-modified electrodes (electrode dsDNA-P1&P2 / AuNPs / GCE, electrode dsDNA-P3&P4 / AuNPs / GCE, electrode dsDNA-P5&P6 / AuNPs / GCE, electrode dsDNA-P7&P8 / AuNPs / GCE) were selected (the concentration ratio of the different dsDNA sequences was 1:1). The peak current of 1 mM L-Trp and 1 mM D-Trp was detected by the DPV method, and the peak current ratio was calculated.

[0058] like Figure 2 As shown, the peak current ratios measured for the four different dsDNA sequences were almost identical, indicating that the dsDNA sequence has almost no effect on the recognition efficiency of tryptophan enantiomers.

[0059] Example 3: Effect of amine structure on tryptophan enantiomer recognition efficiency

[0060] To improve the efficiency of electrode recognition of tryptophan enantiomers, we added ethylamine, diethylamine, and triethylamine to the tryptophan solution. At room temperature, ethylamine, diethylamine, and triethylamine were added to a 1 mM tryptophan PBS solution at an equivalence ratio of 1:1 and mixed continuously for 10 min. This allowed the three amines to form ion-pair complexes with tryptophan, thereby affecting its interaction with chiral recognition sites in DNA.

[0061] from Figure 3 As can be seen, the addition of triethylamine can better improve the recognition efficiency of tryptophan enantiomers by the electrode dsDNA-P1&P2 / AuNPs / GCE. This is because the greater steric hindrance of triethylamine leads to better stereoselectivity of the enantiomer ion-pair complex formed by the DNA pair.

[0062] Example 4: Optimization of Experimental Conditions

[0063] (1) Since DNA itself has chiral recognition sites, which are one of the key factors affecting the enantiomer recognition efficiency, the effect of DNA concentration on the enantiomer recognition efficiency of tryptophan was studied. Since the AuNPs / GCE electrode prepared by gold nanoparticle electrodeposition on the surface of glassy carbon electrode has a stronger response signal to tryptophan than the bare gold electrode, the AuNPs / GCE electrode was used as the working electrode in subsequent experiments.

[0064] like Figure 4As shown, with the increase of dsDNA-P1&P2 concentration, the recognition efficiency of the electrode dsDNA-P1&P2 / AuNPs / GCE for tryptophan enantiomers significantly improved. When the DNA concentration reached 2 μM, the recognition efficiency no longer increased with further increases in DNA concentration, reaching a plateau. This indicates that the DNA immobilized on the electrode reached saturation and could not be immobilized with more DNA. Therefore, this experiment determined the optimal DNA concentration to be 2 μM.

[0065] (2) Since amino acids are protein monomers, their physiological activity is affected by excessively acidic or alkaline environments. Therefore, this experiment investigated the effect of the pH of the tryptophan buffer on the recognition efficiency of tryptophan enantiomers. First, the pH of the PBS buffer was adjusted by changing the ratio of Na2HPO4 to NaH2PO4. Then, tryptophan and triethylamine (equivalent ratio of tryptophan to triethylamine was 1:1) were added to the PBS buffer simultaneously.

[0066] like Figure 5 As shown, within the pH range of 4–7, the recognition efficiency of the electrode dsDNA-P1&P2 / AuNPs / GCE for tryptophan enantiomers gradually increases, reaching its maximum at pH 7 of the buffer solution. Within the pH range of 7–9, the recognition efficiency of the sensor for tryptophan enantiomers gradually decreases. This is because tryptophan's structure is easily destroyed in overly acidic environments, leading to lower recognition efficiency, while in overly alkaline environments, tryptophan denatures, resulting in reduced recognition efficiency. Therefore, this experiment determined the optimal pH value of the buffer solution to be 7.

[0067] (3) Triethylamine and tryptophan form an ion-pair complex, which can affect the recognition efficiency of tryptophan enantiomers by the electrode dsDNA-P1&P2 / AuNPs / GCE. Therefore, this invention also investigated the effect of the binding time of triethylamine and tryptophan and their equivalent ratio on the recognition efficiency of tryptophan enantiomers. Triethylamine was added dropwise to 1 mM tryptophan in PBS buffer (the equivalent ratio of tryptophan to triethylamine was 1:1), and the mixture was shaken continuously to allow triethylamine and tryptophan to be recognized and detected at different binding times.

[0068] Figure 6 The effect of triethylamine binding time to tryptophan on recognition efficiency was shown. It can be seen that the recognition efficiency of the electrode dsDNA-P1&P2 / AuNPs / GCE for tryptophan enantiomers increases with time, reaching a maximum at a binding time of 10 min. Furthermore, the recognition efficiency no longer increases with time and tends to stabilize. Therefore, 10 min was selected as the optimal binding time.

[0069] (4) The effect of different equivalence ratios of tryptophan to triethylamine (TEA) on the recognition efficiency of tryptophan enantiomers.

[0070] from Figure 7 As can be seen, the recognition efficiency gradually increases with the increase of the amount of triethylamine. The recognition efficiency reaches its maximum when the equivalence ratio of tryptophan to triethylamine is 1:1.5. When the amount of triethylamine increases further, the recognition efficiency decreases. This is because the aqueous solution of triethylamine is alkaline. Excess triethylamine will cause the tryptophan buffer to become alkaline, making tryptophan easily denatured and unable to effectively recognize tryptophan enantiomers. Therefore, this experiment selected an optimal equivalence ratio of 1:1.5 for tryptophan enantiomers to triethylamine.

[0071] Note: In Examples 2-4, L-Trp and D-Trp were detected separately to obtain the peak current ratio I of the two tryptophans. L-Trp / I D-Trp The peak current ratio was used to evaluate the electrode's recognition efficiency for tryptophan enantiomers.

[0072] Example 5: Determination of enantiomeric content in a mixture of tryptophan enantiomers

[0073] To verify whether the chiral electrochemical sensor constructed in this experiment can detect the L-Trp and D-Trp contents in the tryptophan enantiomer mixture, solutions containing different percentages of D-Trp (0%, 20%, 40%, 60%, 80%, and 100%) of tryptophan enantiomer mixture were prepared, with a total concentration of 1 mM. The DPV method was used to perform electrochemical detection on these solutions.

[0074] like Figure 8 As shown, with the increase of D-Trp%, the peak current (Ip) gradually decreases, and Ip exhibits a good linear relationship with D-Trp%, with the linear equation being: Ip = -0.067D-Trp% + 21.71(R) 2 =0.9984). The results show that the constructed electrode dsDNA-P1&P2 / AuNPs / GCE can be used to predict the content of L-Trp and D-Trp in tryptophan enantiomer mixtures, which is of great significance for the chiral recognition of tryptophan.

[0075] Example 6: Determination of tryptophan enantiomers based on electrode dsDNA-P1&P2 / AuNPs / GCE

[0076] Under the optimal experimental conditions obtained in Example 4, the constructed electrode dsDNA-P1&P2 / AuNPs / GCE was used to perform DPV detection on different concentrations of tryptophan enantiomers, ranging from 0.1 to 2 mM. The results are as follows: Figure 9As shown: the peak current gradually increases with the increase of L-Trp or D-Trp concentration, and Ip shows a good linear relationship with the concentration of L-Trp or D-Trp. The linear equations for L-Trp and D-Trp are as follows: Ip (L-Trp) =18.67C + 2.71(R) 2 =0.9969) and Ip (D-Trp) =12.78C + 2.07(R) 2 =0.9993). The results indicate that the electrode dsDNA-P1&P2 / AuNPs / GCE has good detection capability for tryptophan enantiomers.

[0077] Example 7: Stability and Selectivity Experiment

[0078] To investigate the stability of the electrode dsDNA-P1&P2 / AuNPs / GCE, we placed the constructed electrode dsDNA-P1&P2 / AuNPs / GCE in a 4°C refrigerator and detected tryptophan enantiomers on days 1, 3, 5, and 7. Figure 10 As shown, its RSD is 0.3%, indicating that the electrode dsDNA / AuNPs / GCE has good stability.

[0079] To further investigate the selectivity of the electrode dsDNA-P1&P2 / AuNPs / GCE, we used the electrode dsDNA-P1&P2 / AuNPs / GCE and an automated polarimeter to detect racemic solutions of tryptophan containing other amino acids. Figure 11 The results showed that adding 10 mM of L-Phe, L-Lys, Gly, L-Asp, and KNO3 to a 1 mM racemic solution of tryptophan (50% L-Trp and 50% D-Trp) and performing DPV analysis revealed that these compounds had almost no interference with the peak current magnitude, with an RSD of 0.5%. Figure 12 The results showed that adding 10 mM L-Phe, L-Lys, and L-Asp to a 10 mM racemic solution of tryptophan (50% L-Trp and 50% D-Trp) and measuring their optical rotation revealed significant changes in optical rotation upon the addition of other amino acids. This indicates that our constructed electrode dsDNA / AuNPs / GCE exhibits higher selectivity than the classic polarimetric method.

[0080] Example 8: Sample Determination Experiment

[0081] Two samples containing different impurities were prepared: one was a mixture of tryptophan enantiomers containing different proportions of L-Trp and D-Trp with 10% KNO3 added; the other was a mixture of tryptophan enantiomers containing different proportions of L-Trp and D-Trp with 2.5% L-Lys, 5% Gly, and 2.5% L-Asp added. Solutions containing only L-Trp, only D-Trp, and the two impurity-containing samples were prepared to the same concentration, diluted, and their absorbance was measured at 279 nm using ultraviolet spectrophotometry. 1 The concentration is calculated according to Beer-Lambert Law. The concentration of the solution before dilution is obtained according to the dilution factor. Then the concentration of the solution before dilution is diluted to 1mM.

[0082] The electrode dsDNA-P1&P2 / AuNPs / GCE was inserted into PBS buffer containing the above sample and triethylamine. The peak current was measured using the DPV method, based on the linear equation for tryptophan content (Ip = -0.067D-Trp% + 21.71, R...). 2 =0.9984, Figure 8 A) The ratios of L-Trp and D-Trp were obtained, and the results are shown in Tables 1 and 2.

[0083] Table 1 shows the detection of D-Trp and L-Trp tryptophan content in the samples.

[0084]

[0085] In Table 1, Sample 1 contains 12% D-Trp and 88% L-Trp, totaling 450.0 mg, with 10% KNO3 (50.0 mg) added; Sample 2 contains 45% D-Trp and 55% L-Trp, totaling 450.0 mg, with 10% KNO3 (50.0 mg) added; Sample 3 contains 88% D-Trp and 12% L-Trp, totaling 450.0 mg, with 10% KNO3 (50.0 mg) added.

[0086] Table 2 shows the detection of D-Trp and L-Trp tryptophan content in the samples.

[0087]

[0088] In Table 2, sample 4 contained 12% D-Trp and 88% L-Trp, totaling 180.0 mg, and added 2.5% L-Lys, 5% Gly, and 2.5% L-Asp, totaling 20.0 mg; sample 5 contained 45% D-Trp and 55% L-Trp, totaling 180.0 mg, and added 2.5% L-Lys, 5% Gly, and 2.5% L-Asp, totaling 20.0 mg; sample 6 contained 88% D-Trp and 12% L-Trp, totaling 180.0 mg, and added 2.5% L-Lys, 5% Gly, and 2.5% L-Asp, totaling 20.0 mg.

[0089] The above results show that the ratio of L-Trp to D-Trp can be predicted regardless of whether inorganic salts or other chiral amino acids are present. This fully demonstrates that the chiral electrochemical sensor provided by this invention can successfully predict the percentage content of two tryptophan enantiomers in a sample, which is of great significance in practical applications.

[0090] This invention provides a method for detecting tryptophan enantiomers using a chiral electrochemical sensor constructed from ion-pair interactions. First, an appropriate compound is selected to form an ion pair with the tryptophan enantiomer, thereby altering the spatial stereostructure of the tryptophan enantiomer and enabling the chiral recognition site on the dsDNA at the sensing interface to better and more effectively recognize the tryptophan enantiomer. This invention optimizes factors affecting the recognition efficiency of the electrode dsDNA / AuNPs / GCE, such as DNA concentration, pH, and the binding time and equivalence ratio of triethylamine to tryptophan. Under optimal conditions, the peak current ratio (IL) of L-Trp and D-Trp is detected by differential pulse voltammetry (DPV). L-Trp / I D-Trp The coefficient of performance (COP) was 1.45, and L-Trp and D-Trp exhibited a good linear relationship within the concentration range of 0.1–2 mM. The sensor also demonstrated good stability and anti-interference capabilities. Furthermore, sensitive determination of the L-Trp and D-Trp ratio in unknown real samples was achieved using ultraviolet spectrophotometry and electrochemical methods. This invention provides a new approach for constructing electrochemical chiral sensors, demonstrating their significant potential in chiral recognition applications.

[0091] References:

[0092] 1、QMYe,LLGuo,DTWu,BZYang,YXTao,LHDeng,Y.Kong.Covalentfunctionalization of bovine serum albumin with graphene quantum dots forstereospecific molecular recognition.Analytical Chemistry,2019;91(186):1871-11111.

Claims

1. The method of detecting tryptophan enantiomers using a chiral electrochemical sensor constructed by ion-pair interactions utilizes DNA as a chiral selector. Based on the fact that the ion pairs formed by the two tryptophan enantiomers and amine compounds have different binding abilities to DNA, the peak current signals of the two tryptophan enantiomers in the voltammetry diagram will show differences, thereby realizing the identification and detection of tryptophan enantiomers. The specific operating steps are as follows: Insert the electrode dsDNA / AuNPs / GCE into a PBS buffer containing tryptophan enantiomers and amine compounds, and perform electrochemical detection using differential pulse voltammetry, with a scan potential of 0.4~1.1V and a pulse amplitude of 50 mVs. –1 The pulse width is 0.05 s, and the peak current ratio is used to evaluate the recognition efficiency. In the above operation steps, the amine compound in the PBS buffer containing the tryptophan enantiomer and the amine compound is any one of ethylamine, diethylamine, or triethylamine.

2. The method for detecting tryptophan enantiomers using a chiral electrochemical sensor constructed from ion-pair interactions according to claim 1, characterized in that: The amine compound mentioned is triethylamine.

3. The method for detecting tryptophan enantiomers using a chiral electrochemical sensor constructed from ion-pair interactions according to claim 1, characterized in that: The equivalent ratio of the tryptophan enantiomer to the amine compound is 1:0.1 to 1:2.

5.

4. The method for detecting tryptophan enantiomers using a chiral electrochemical sensor constructed based on ion-pair interactions according to claim 3, characterized in that: The equivalence ratio of tryptophan enantiomers to amine compounds is 1:1.

5.

5. The method for detecting tryptophan enantiomers using a chiral electrochemical sensor constructed from ion-pair interactions according to claim 2, characterized in that: The equivalent ratio of tryptophan enantiomer to triethylamine is 1:1.

5.

6. The method for detecting tryptophan enantiomers using a chiral electrochemical sensor constructed from ion-pair interactions according to claim 1, characterized in that: The PBS buffer is a mixed solution of 10 mM Na2HPO4, 10 mM NaH2PO4 and 0.1 M KCl, with a pH of 7.0.

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