Method for detecting cysteine homolog based on Raman spectrum

Abstract

A method for detecting cysteine homologues based on Raman spectroscopy comprises the following steps: firstly, preparing cysteine homologue standard substance solutions, 2, 3-naphthalenedicarboxaldehyde solutions and buffer solutions with different concentrations, and then carrying out derivatization treatment on the cysteine homologue standard substance solutions to obtain cysteine homologue standard substance derivative solutions; combining the SERS substrate with the cysteine homolog standard substance derivative solution to perform signal enhancement, and then performing Raman spectrum detection to obtain the Raman spectrum of the cysteine homolog standard substance derivative solution; and finally, drawing a Raman spectrum standard curve of the cysteine homolog standard substance derivative liquid, and obtaining a cysteine homolog working curve equation and related coefficients according to the determined cysteine homolog spectrum characteristic peak intensity and the cysteine homolog standard substance derivative liquid concentration. According to the method, qualitative and quantitative analysis of the cysteine homologues can be simply and quickly realized, the operation is simple, the sensitivity is high, and the method has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of compound detection, and in particular to a method for detecting cysteine homologues based on Raman spectroscopy. Background Art

[0002] Cysteine and its homologue, homocysteine, have attracted significant attention in the food and medical fields. Cysteine deficiency can lead to symptoms such as hair loss, lethargy, liver damage, skin lesions, and muscle and fat loss, while elevated cysteine levels can contribute to Alzheimer's disease and cardiovascular disease. Homocystine, a homologue of cysteine, contains an additional methylene group before the thiol group in the side chain. Homocystine is an important biomarker for numerous diseases and a key essential amino acid found in human blood. It is also a sulfur-containing amino acid involved in energy metabolism and various methylation reactions. Elevated homocysteine levels in serum may contribute to cognitive decline and dementia. Cysteine and homocysteine are closely linked, and many pathological conditions, including Alzheimer's disease, Parkinson's disease, and autoimmune deficiency syndromes, are associated with elevated levels of both. Therefore, the ability to simultaneously monitor cysteine and homocysteine levels is crucial.

[0003] Currently, methods for detecting cysteine and its homologues include high-performance liquid chromatography, fluorescence, UV-visible spectroscopy, and electrochemical methods. However, methods that can simultaneously detect cysteine and its homologues are extremely rare. Summary of the Invention

[0004] The technical problem solved by the present invention is to provide a method for detecting cysteine homologues based on Raman spectroscopy to solve the problems in the above-mentioned background technology.

[0005] The technical problem solved by the present invention is achieved by adopting the following technical solutions: A method for detecting cysteine homologues based on Raman spectroscopy, comprising the following steps: (1) Preparation of cysteine standard solution First prepare 10 -2 Prepare 10 M cysteine stock solution by weighing -2 The mass of the solid cysteine sample required to prepare a cysteine solution of 10 M was then dissolved in methanol to obtain a 10 -2 M cysteine stock solution, and then use ultrapure water to dilute the prepared cysteine stock solution step by step to a concentration of 10 -4 M, 10 -6 M, 10 -8 M, 10 -10 M and 10 -12M cysteine standard solution; (2) Preparation of homocysteine standard solution First prepare 10 -2 Prepare 10 M homocysteine stock solution by weighing -2 The mass of homocysteine solid sample required to prepare homocysteine solution of 10 M was calculated, and then methanol was added to the homocysteine solid sample to dissolve it and obtain a homocysteine solution with a concentration of 10 -2 M homocysteine stock solution; then use ultrapure water to dilute the prepared homocysteine stock solution step by step to a concentration of 10 -4 M, 10 -6 M, 10 -8 M, 10 -10 M and 10 -12 M homocysteine standard solution; (3) Preparation of 2,3-naphthalene dicarboxaldehyde solution Weigh out 10 -2 The mass of the 2,3-naphthalene dicarboxaldehyde solid sample required for the 2,3-naphthalene dicarboxaldehyde solution was calculated, and then methanol was added to the 2,3-naphthalene dicarboxaldehyde solid sample to dissolve it and obtain a concentration of 10 -2 M 2,3-naphthalene dicarboxaldehyde solution, and then use ultrapure water to 10 -2 The 2,3-naphthalene dicarboxaldehyde solution was diluted to 10 -4 M 2,3-naphthalene dicarboxaldehyde solution; (4) Preparation of borax-sodium hydroxide buffer solution First, prepare 0.05 M sodium tetraborate solution and 0.2 M sodium hydroxide solution, respectively. Then, mix the sodium tetraborate solution and sodium hydroxide solution in a volume ratio of 50:6-46 and dilute to 200 mL to obtain a borax-sodium hydroxide buffer solution with a pH of 9.3-10.1. (5) Derivatization of cysteine standard solution The borax-sodium hydroxide buffer solution with a pH of 9.8, the cysteine standard solution prepared in step (1), and the 2,3-naphthalene dicarboxaldehyde solution prepared in step (3) are sequentially added to the reactor and mixed, and the volume ratio of the added borax-sodium hydroxide buffer solution, the cysteine standard solution, and the 2,3-naphthalene dicarboxaldehyde solution is 1:1:1. The derivatization reaction time is 5-100 min under magnetic stirring at a temperature of 0-100°C, and the mixture is naturally cooled to room temperature to obtain a cysteine standard derivative solution; (6) Derivatization of homocysteine standard solution The borax-sodium hydroxide buffer solution with a pH of 9.8, the homocysteine standard solution prepared in step (2), and the 2,3-naphthalene dicarboxaldehyde solution prepared in step (3) are sequentially added to the reactor and mixed, and the volume ratio of the added borax-sodium hydroxide buffer solution, the homocysteine standard solution, and the 2,3-naphthalene dicarboxaldehyde solution is 1:1:1. The derivatization reaction time is 5-100 min under magnetic stirring at a temperature of 0-100°C, and the mixture is naturally cooled to room temperature to obtain a homocysteine standard derivative solution; (7) Raman spectroscopy detection of cysteine standard derivative solution The SERS substrate is combined with the cysteine standard derivative solution obtained in step (5) to perform signal enhancement, and then Raman spectroscopy detection is performed to obtain a Raman spectrum of the cysteine standard derivative solution; (8) Raman spectroscopy detection of homocysteine standard derivative solution The SERS substrate is combined with the homocysteine standard derivative solution obtained in step (6) to perform signal enhancement, and then Raman spectroscopy is performed to obtain a Raman spectrum of the homocysteine standard derivative solution; (9) Draw the Raman spectrum standard curve of cysteine standard derivative solution The concentration of cysteine standard derivatization solution is used as the horizontal axis. Cysteine is located at 1377 cm -1 The Raman characteristic peak intensity of cysteine was used as the vertical axis to draw the Raman spectrum standard curve of the cysteine standard derivative solution. -1 The cysteine working curve equation and correlation coefficient are obtained by combining the intensity of the cysteine Raman characteristic peak determined by Raman characteristic peak and the concentration of the cysteine standard derivatization solution; (10) Draw the Raman spectrum standard curve of homocysteine standard derivative solution The concentration of homocysteine standard derivatization solution was used as the horizontal axis, and homocysteine was selected at 665 cm -1 The Raman characteristic peak intensity is used as the vertical axis to draw the Raman spectrum standard curve of the homocysteine standard derivative solution. -1 The homocysteine working curve equation and correlation coefficient were obtained by combining the intensity of the homocysteine Raman characteristic peak determined by Raman characteristic peak and the concentration of the homocysteine standard derivatization solution.

[0006] Furthermore, in step (6) or step (7), the SERS substrate is made of nanomaterials such as silver.

[0007] Furthermore, in step (9), the cysteine working curve is cysteine based on 1377 cm -1There is a linear relationship between the SERS intensity (I) of the characteristic peak and the logarithm of the cysteine concentration (LgC). The cysteine working curve equation is I=4662.48+354.32LgC, and the correlation coefficient is R 2 The detection limit was 1.5×10 -12 M.

[0008] Furthermore, in step (10), the homocysteine working curve is homocysteine based on 665 cm -1 There is a linear relationship between the intensity of the Raman characteristic peak (I) and the logarithmic value of the homocysteine concentration (LgC). The working curve equation for homocysteine is I = 5070.14 + 406.45LgC, the correlation coefficient R² is 0.9971, and the detection limit is 1.5×10 -12 M. Beneficial effects

[0009] 1. The present invention uses 2,3-naphthalene dicarboxaldehyde to carry out a derivatization reaction with a cysteine (or homocysteine) standard solution, effectively improving the affinity of cysteine (or homocysteine) with the SERS substrate. The derivatized solution is then directly subjected to surface-enhanced Raman spectroscopy detection and analysis, thereby simplifying the operation steps and reducing the detection limit of cysteine (or homocysteine); 2. The present invention can selectively detect cysteine and its homologue homocysteine, two organic molecules with very similar structures, by adjusting the derivatization reaction conditions; 3. The present invention can simply and quickly realize the qualitative and quantitative analysis of trace cysteine and homocysteine; 5. The detection limit of cysteine and homocysteine in the present invention is 1.5×10 -12 M, simple operation, high sensitivity, and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of surface-enhanced Raman spectroscopy of cysteine derivative solution in Example 1 of the present invention.

[0011] Figure 2 Schematic diagram of surface-enhanced Raman spectroscopy of cysteine standard derivative solutions with different concentrations in Example 1 of the present invention.

[0012] Figure 3 Schematic diagram of the Raman spectrum standard curve of the cysteine standard derivative solution in Example 1 of the present invention.

[0013] Figure 4 Schematic diagram of surface-enhanced Raman spectroscopy of the homocysteine standard derivative solution in Example 2 of the present invention.

[0014] Figure 5 Schematic diagram of surface-enhanced Raman spectroscopy of homocysteine standard derivative solutions with different concentrations in Example 2 of the present invention.

[0015] Figure 6 Schematic diagram of the Raman spectrum standard curve of the homocysteine standard derivative solution in Example 2 of the present invention. DETAILED DESCRIPTION

[0016] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below with reference to specific illustrations. Example

[0017] A method for rapid detection of cysteine, the specific steps are as follows: (1) Prepare different concentrations of cysteine standard solution and 2,3-naphthalene dicarboxaldehyde solution buffer A. Preparation of cysteine standard solution First prepare 10 -2 Prepare 10 M cysteine stock solution by weighing -2 The mass of the solid cysteine sample required to prepare a cysteine solution of 10 M was then dissolved in methanol to obtain a 10 -2 M cysteine stock solution, and then use ultrapure water to dilute the prepared cysteine stock solution step by step to a concentration of 10 -4 M, 10 -6 M, 10 -8 M, 10 -10 M and 10 -12 M cysteine standard solution; B. Preparation of 2,3-naphthalene dicarboxaldehyde solution Weigh out 10 -2 The mass of the 2,3-naphthalene dicarboxaldehyde solid sample required for the 2,3-naphthalene dicarboxaldehyde solution was calculated, and then methanol was added to the 2,3-naphthalene dicarboxaldehyde solid sample to dissolve it and obtain a concentration of 10 -2 M 2,3-naphthalene dicarboxaldehyde solution, and then use ultrapure water to 10 -2 The 2,3-naphthalene dicarboxaldehyde solution was diluted to 10 -4 M 2,3-naphthalene dicarboxaldehyde solution; (2) Derivatization treatment of cysteine standard solution The pH 9.8 borax-sodium hydroxide buffer solution, the cysteine standard solution prepared in step (1), and the 2,3-naphthalene dicarboxaldehyde solution were sequentially added to the reactor and mixed, and the volume ratio of the added borax-sodium hydroxide buffer solution, the cysteine standard solution, and the 2,3-naphthalene dicarboxaldehyde solution was 1:1:1. The derivatization reaction time was 60 min under magnetic stirring at a temperature of 25°C, and the mixture was naturally cooled to room temperature to obtain a cysteine standard derivative solution. (3) Raman spectroscopy detection of cysteine standard derivative solution The SERS substrate is combined with the cysteine standard derivative solution obtained in step (2) to enhance the signal, and then Raman spectroscopy is performed to detect the Raman spectrum of the cysteine standard derivative solution. Figure 1 As shown; The Raman spectroscopy test parameters are as follows: The laser wavelength is fixed at 785 nm, the laser energy is 5 mW, and the instrument resolution is accurate to 5 cm -1 The exposure time for a single acquisition was 2 s, the number of sample exposures was 2, the slit was 25 μm, and the grating was 400 lines / mm. Under the above Raman spectroscopy test parameters, the cysteine standard derivatization solution was analyzed and detected. (4) Draw the Raman spectrum standard curve of cysteine standard derivative solution Prepare different concentrations of cysteine standard derivative solutions according to the steps in step (2). Mix 20 mL of borax-sodium hydroxide buffer (pH 9.8) with different concentrations of cysteine standard solutions (10 -4 M, 10 -6 M, 10 -8 M, 10 -10 M and 10 -12 M) and 2,3-naphthalene dicarboxaldehyde solution (10 -4 M) was added to the reactor for mixing, and the volume ratio of the added borax-sodium hydroxide buffer solution, cysteine standard solution and 2,3-naphthalene dicarboxaldehyde solution was 1:1:1. The mixture was reacted for 60 min under magnetic stirring at 25°C. After naturally cooling to room temperature, cysteine standard derivative solutions of different concentrations were obtained. The SERS substrate was then combined with the cysteine standard derivative solutions of different concentrations for signal enhancement. The Raman spectrum test parameters in step (3) were used for detection, and the Raman spectra of the cysteine standard derivative solutions of different concentrations were obtained as shown in FIG. Figure 2 As shown; the concentration of cysteine standard derivatization solution is used as the horizontal axis, cysteine 1377 cm -1 The Raman characteristic peak intensity is used as the vertical axis to draw the Raman spectrum standard curve of the cysteine standard derivative solution, as shown in Figure 3 As shown, the linear range is 10 -12~10 -4 M, from cysteine 1377 cm -1 The intensity of the cysteine Raman characteristic peak measured at the Raman characteristic peak and the concentration of the cysteine standard derivative solution were used to obtain the cysteine working curve equation I=354.32LgC+4662.48, and the correlation coefficient R 2 The detection limit was 1.5×10 -12 M. Example

[0018] A method for rapid detection of homocysteine, comprising the following steps: (1) Prepare homocysteine standard solutions and 2,3-naphthalene dicarboxaldehyde solution buffer solutions of different concentrations I. Preparation of homocysteine standard solution First prepare 10 -2 Prepare 10 M homocysteine stock solution by weighing -2 The mass of homocysteine solid sample required to prepare homocysteine solution of 10 M was calculated, and then methanol was added to the homocysteine solid sample to dissolve it and obtain a homocysteine solution with a concentration of 10 -2 M homocysteine stock solution; then use ultrapure water to dilute the prepared homocysteine stock solution step by step to a concentration of 10 -4 M, 10 -6 M, 10 -8 M, 10 -10 M and 10 -12 M homocysteine standard solution; II. Preparation of 2,3-naphthalene dicarboxaldehyde solution Weigh out 10 -2 The mass of the 2,3-naphthalene dicarboxaldehyde solid sample required for the 2,3-naphthalene dicarboxaldehyde solution was calculated, and then methanol was added to the 2,3-naphthalene dicarboxaldehyde solid sample to dissolve it and obtain a concentration of 10 -2 M 2,3-naphthalene dicarboxaldehyde solution, and then use ultrapure water to -2 The 2,3-naphthalene dicarboxaldehyde solution was diluted to 10 -4 M 2,3-naphthalene dicarboxaldehyde solution; (2) Derivatization treatment of homocysteine standard solution The pH 9.8 borax-sodium hydroxide buffer solution, the homocysteine standard solution prepared in step (1), and the 2,3-naphthalene dicarboxaldehyde solution were sequentially added to the reactor and mixed, and the volume ratio of the added borax-sodium hydroxide buffer solution, the homocysteine standard solution, and the 2,3-naphthalene dicarboxaldehyde solution was 1:1:1. The derivatization reaction time was 40 min under magnetic stirring at a temperature of 75°C, and the mixture was naturally cooled to room temperature to obtain a homocysteine standard derivative solution; (3) Raman spectroscopy detection of homocysteine standard derivative solution The SERS substrate is combined with the homocysteine standard derivative solution prepared in step (2) to enhance the signal, and then Raman spectroscopy is performed to detect the Raman spectrum of the homocysteine standard derivative solution. Figure 4 As shown; The Raman spectroscopy test parameters are as follows: The laser wavelength is fixed at 785 nm, the laser energy is 5 mW, and the instrument resolution is accurate to 5 cm -1 The exposure time for a single acquisition was 2 s, the number of sample exposures was 2, the slit was 25 μm, and the grating was 400 lines / mm. Under the above Raman spectroscopy test parameters, the homocysteine standard derivatization solution was analyzed and detected; (4) Raman spectral standard curve of homocysteine standard derivative solution According to the steps of step (2), homocysteine standard solutions of different concentrations were prepared. 20 mL of borax-sodium hydroxide buffer (pH 9.8) was mixed with the homocysteine standard solutions of different concentrations prepared in step (1) (10 -4 M, 10 -6 M, 10 -8 M, 10 -10 M and 10 -12 M) and 2,3-naphthalene dicarboxaldehyde solution (10 -4 M) was added to the reactor for mixing, and the volume ratio of the added borax-sodium hydroxide buffer solution, homocysteine standard solution and 2,3-naphthalene dicarboxaldehyde solution was 1:1:1. The mixture was reacted under magnetic stirring at 75°C for 40 min. After naturally cooling to room temperature, homocysteine standard solutions of different concentrations were obtained. The SERS substrate was then combined with the obtained homocysteine standard derivative solutions of different concentrations for signal enhancement. The Raman spectrum test parameters described in step (3) were used for detection. The obtained Raman spectrum is shown in FIG. Figure 5 As shown; the concentration of homocysteine standard derivatization solution is used as the horizontal axis, homocysteine 665 cm -1 The Raman characteristic peak intensity of is used as the vertical axis to draw the Raman spectrum standard curve of the homocysteine standard derivative solution, as shown in Figure 6 As shown, the linear range is 10 -12 ~10 -4 M, composed of homocysteine 665 cm -1 The intensity of the homocysteine Raman characteristic peak measured at the Raman characteristic peak was compared with the concentration of the homocysteine standard derivatization solution to obtain the homocysteine working curve equation I = 406.45lgC + 5070.14, with a correlation coefficient R² = 0.9971 and a detection limit of 1.5×10-12 M.

[0019] In the above-mentioned Example 1 to Example 2, the borax-sodium hydroxide buffer solution was prepared as follows: First, prepare 0.05 M sodium tetraborate solution (X) and 0.2 M sodium hydroxide solution (Y), respectively. Then, mix the prepared sodium tetraborate solution and sodium hydroxide solution according to the ratio in Table 1 and adjust the volume to 200 mL to obtain a borax-sodium hydroxide buffer solution with a pH of 9.3-10.1.

[0020] Table 1 Borax-sodium hydroxide buffer ratio

[0021] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for detecting cysteine homologues based on Raman spectroscopy, characterized in that: The specific steps are as follows: (1) Prepare different concentrations of cysteine homologue standard solution, 2,3-naphthalene dicarboxaldehyde solution and buffer solution respectively; (2) Derivatizing the cysteine homologue standard solution, sequentially adding a buffer solution with a certain volume ratio, a cysteine homologue standard solution of different concentrations, and a 2,3-naphthalene dicarboxaldehyde solution into a reactor and mixing them for derivatization to obtain a cysteine homologue standard derivative solution; (3) performing Raman spectroscopy detection on the cysteine homologue standard derivative solution, combining the SERS substrate with the cysteine homologue standard derivative solution obtained in step (2) to perform signal enhancement, and then performing Raman spectroscopy detection to obtain a Raman spectrum of the cysteine homologue standard derivative solution; (4) Draw a Raman spectrum standard curve of the cysteine homologue standard derivative solution, with the concentration of the cysteine homologue standard derivative solution as the horizontal axis and the intensity of the characteristic peak of the Raman spectrum of the cysteine homologue standard derivative solution as the vertical axis, and draw a Raman spectrum standard curve of the cysteine homologue standard derivative solution; Then, the working curve equation and correlation coefficient of the cysteine homologues are obtained based on the Raman characteristic intensity of the cysteine homologues measured at the Raman characteristic peak of the cysteine homologues and the concentration of the cysteine homologue standard derivatization solution.

2. The method for detecting cysteine homologues based on Raman spectroscopy according to claim 1, characterized in that: In step (1), different concentrations of cysteine homologue standard solutions are prepared as follows: First prepare 10 -2 Weigh 10 M of cysteine homologue stock solution to prepare -2 The mass of the solid sample of cysteine homologues required for the cysteine homologue solution was then added with methanol to dissolve the solid sample of cysteine homologues to obtain a concentration of 10 -2 M cysteine homologue stock solution, and then use ultrapure water to dilute the prepared cysteine homologue stock solution step by step to a concentration of 10 -4 M, 10 -6 M, 10 -8 M, 10 -10 M and 10 -12 M cysteine homologue standard solution.

3. The method for detecting cysteine homologues based on Raman spectroscopy according to claim 1, characterized in that: In step (1), the 2,3-naphthalene dicarboxaldehyde solution is prepared as follows: Weigh out 10 -2 The mass of the 2,3-naphthalene dicarboxaldehyde solid sample required for the 2,3-naphthalene dicarboxaldehyde solution was calculated, and then methanol was added to the 2,3-naphthalene dicarboxaldehyde solid sample to dissolve it and obtain a concentration of 10 -2 M 2,3-naphthalene dicarboxaldehyde solution, and then use ultrapure water to 10 -2 The 2,3-naphthalene dicarboxaldehyde solution was diluted to 10 -4 M of 2,3-naphthalene dicarboxaldehyde solution.

4. The method for detecting cysteine homologues based on Raman spectroscopy according to claim 1, characterized in that: In step (1), the buffer solution is a borax-sodium hydroxide buffer solution.

5. The method for detecting cysteine homologues based on Raman spectroscopy according to claim 4, characterized in that: Prepare borax-sodium hydroxide buffer as follows: First, 0.05 M sodium tetraborate solution and 0.2 M sodium hydroxide solution were prepared separately. Then, the sodium tetraborate solution and sodium hydroxide solution were mixed in a volume ratio of 50:6~50 and the volume was adjusted to 200 mL to obtain a borax-sodium hydroxide buffer solution with a pH of 9.3~10.

1.

6. The method for detecting cysteine homologues based on Raman spectroscopy according to claim 1, characterized in that: In step (2), the derivatization treatment of the cysteine homologue standard solution is specifically as follows: Borax-sodium hydroxide buffer, the cysteine homologue standard solution prepared in step (1) and the 2,3-naphthalene dicarboxaldehyde solution are sequentially added to the reactor and mixed, and the volume ratio of the added borax-sodium hydroxide buffer, the cysteine standard solution and the 2,3-naphthalene dicarboxaldehyde solution is 1:1:

1. The derivatization reaction time is 5-100 min under magnetic stirring at a temperature of 0-100°C, and the mixture is naturally cooled to room temperature to obtain a cysteine homologue standard derivative solution.

7. The method for detecting cysteine homologues based on Raman spectroscopy according to claim 1, characterized in that: In step (3), the SERS substrate is made of nanomaterials such as silver.

8. The method for detecting cysteine homologues based on Raman spectroscopy according to claim 1, characterized in that: The cysteine homologues include cysteine and homocysteine.

9. The method for detecting cysteine homologues based on Raman spectroscopy according to claim 8, wherein: The cysteine working curve is based on 1377 cm -1 There is a linear relationship between the SERS intensity (I) of the Raman characteristic peak and the logarithm of the concentration of the cysteine standard derivative solution (LgC). The cysteine working curve equation is I=4662.48+354.32LgC, and the correlation coefficient is R 2 The detection limit was 1.5×10 -12 M.

10. The method for detecting cysteine homologues based on Raman spectroscopy according to claim 8, wherein: The homocysteine working curve is based on the homocysteine at 665 cm -1 There is a linear relationship between the intensity of the Raman characteristic peak (I) and the logarithmic value of the concentration of the homocysteine standard derivative solution (LgC). The working curve equation for homocysteine is I = 5070.14 + 406.45LgC, the correlation coefficient R² is 0.9971, and the detection limit is 1.5×10 -12 M.

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