Method for detecting L-cysteine in serum based on N, S co-doped carbon nanodots

Abstract

The invention discloses a method for detecting L-cysteine in serum based on N, S co-doped carbon nanodots, and belongs to the field of analysis and detection. The method comprises the following steps: (1) preparing carbon dots by taking beta-mercaptoethylamine and p-phenylenediamine as precursors; (2) after Fe < 3 + > is added into the carbon dot solution, a carbon dot fluorescence quenching phenomenon occurs, and a carbon dot-Fe < 3 + > mixed system is obtained; then adding L-cysteine into the carbon dot-Fe < 3 + > mixed system to gradually recover the fluorescence of the system; and (3) constructing a linear relationship between the concentration of the L-cysteine and the fluorescence recovery degree to obtain the quantitative detection model for the L-cysteine in the serum sample. According to the method, the carbon dots are prepared by taking beta-mercaptoethylamine and p-phenylenediamine as precursors for the first time, quantitative detection of L-cysteine is realized, and the method is low in cost, high in sensitivity, suitable for field rapid detection and relatively high in practical value and popularization prospect.

Description

Technical Field

[0001] The invention relates to a method for detecting L-cysteine ​​in serum based on N and S co-doped carbon nanodots, belonging to the field of analysis and detection. Background Art

[0002] Cysteine ​​(Cys) is the only amino acid among the 20 common amino acids that contains a free sulfhydryl group and is an important amino acid in the human body. Whether the level of cysteine ​​changes normally has a significant impact on human body function. Abnormal cysteine ​​levels can lead to many diseases, such as increased risk of cardiovascular and cerebrovascular diseases, endothelial cell damage, vascular occlusion, and nerve damage2. When the concentration of L-cysteine ​​and cystine in urine is too high (usually up to 100μM), hepatic cystinuria, a genetic disease of amino acid transport, occurs. Therefore, the detection of L-cysteine ​​is of great significance for monitoring human health.

[0003] At present, the common methods for detecting L-cysteine ​​are: chromatography, mass spectrometry and electrochemical voltammetry. Among them, although chromatography and mass spectrometry have high sensitivity and high accuracy, the equipment is expensive and the operation is complicated, which is not suitable for on-site detection, such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), etc. Electrochemical voltammetry is relatively simple, but faces the problem of insufficient sensitivity, especially in the detection of low concentrations of the analyte.

[0004] As a new type of fluorescent carbon nanomaterial, carbon dots have the characteristics of high stability, low cost, easy preparation and convenient use. The prior art "Study on the application of fluorescence "off-on" strategy in the detection of thiol small molecules" reported a detection system using carbon dots in combination with quencher silver nanoparticles (AgNPs). However, when relying on this detection system to detect L-cysteine, it also has a significant response to glutathione (GSH), homocysteine ​​(Hcy), etc., and cannot achieve specific detection of L-cysteine.

[0005] Therefore, there is an urgent need to develop a simple, efficient, sensitive, wide-range, and specific method for detecting L-cysteine. Summary of the invention

[0006] Technical issues:

[0007] Among the existing L-cysteine ​​detection technologies, chromatography and mass spectrometry have high accuracy, but they are expensive, complicated to operate, and unsuitable for rapid on-site detection. Although electrochemical voltammetry is simpler, it is not sensitive enough, especially in the detection of low-concentration L-cysteine, and it is difficult to meet the needs of efficient and low-cost detection. In addition, the existing carbon dot fluorescence detection has a limited detection range and cannot specifically detect L-cysteine.

[0008] Technical solution:

[0009] The present invention provides a method for detecting L-cysteine ​​in serum based on N and S co-doped carbon nanodots, comprising the following steps:

[0010] (1) Using β-mercaptoethylamine and p-phenylenediamine as precursors, carbon dots were obtained through hydrothermal reaction;

[0011] (2) The obtained carbon dots are first mixed with a trivalent iron salt solution to form a carbon dot-Fe 3+ A mixed system solution is then added with a series of L-cysteine ​​solutions of known concentrations, and the mixture is incubated for a period of time. The fluorescence intensity before and after the addition of L-cysteine ​​is then detected by fluorescence spectroscopy to obtain the corresponding fluorescence intensity change value, i.e., the fluorescence recovery degree;

[0012] (3) Using the fluorescence recovery degree and Fe 3+ The quantitative detection model was constructed based on ion concentration.

[0013] In one embodiment of the present invention, the process of synthesizing carbon dots in step (1) includes: mixing β-mercaptoethylamine, p-phenylenediamine and deionized water, and performing a hydrothermal reaction after fully dissolving; after the reaction is completed, purifying by a chromatography column, then concentrating, and diluting with an aqueous solution to obtain a carbon dot solution.

[0014] In one embodiment of the present invention, the mass ratio of β-mercaptoethylamine to p-phenylenediamine is 1:(1.0-2.0).

[0015] In one embodiment of the present invention, the temperature of the hydrothermal reaction is 150-200°C, and specifically 180°C.

[0016] In one embodiment of the present invention, the hydrothermal reaction time is 5-15 hours, and specifically 8 hours.

[0017] In one embodiment of the present invention, the aqueous solution is diluted 100-400 times.

[0018] In one embodiment of the present invention, the process of synthesizing carbon dots in step (1) specifically includes: weighing 0.0771 g of β-mercaptoethylamine and 0.1081 g of p-phenylenediamine in a beaker, adding 25 mL of deionized water, ultrasonically treating for 10 minutes, and transferring the mixture into a 50 mL polytetrafluoroethylene liner after fully dissolving it, and reacting it at 180° C. for 8 hours.

[0019] In one embodiment of the present invention, the eluents used for purification by chromatography column are methanol and ethyl acetate, and the solution emitting green light is extracted.

[0020] In one embodiment of the present invention, the volume ratio of methanol to ethyl acetate is 1:10.

[0021] In one embodiment of the present invention, the silica powder used in the chromatography column is 300-400 mesh.

[0022] In one embodiment of the present invention, step (1) further comprises: concentrating the purified solution, using a small amount of anhydrous ethanol to dissolve the carbon dots remaining on the spherical bottle after concentration, and finally diluting it with an aqueous solution to obtain a carbon dot solution.

[0023] In one embodiment of the present invention, in step (2), the concentration of the ferric salt solution is 100-500 μM, and specifically 200 μM.

[0024] In one embodiment of the present invention, in step (2), the trivalent iron salt is ferric chloride.

[0025] In one embodiment of the present invention, in step (2), the volume ratio of the carbon dot solution to the trivalent iron salt solution is 1:1.

[0026] In one embodiment of the present invention, in step (2), L-cysteine ​​solution and carbon dot-Fe 3+ The volume ratio of the mixed system solution is 1:2.

[0027] In one embodiment of the present invention, in step (2), the incubation time is 0.5-2 hours, and specifically 1 hour.

[0028] In one embodiment of the present invention, in step (2), the concentration of the L-cysteine ​​solution is 0 to 300 μM.

[0029] In one embodiment of the present invention, in step (2), the conditions for fluorescence spectrum detection are: the fluorescence spectrum is measured using a fluorescence spectrometer, the excitation slit width of the spectrometer is 2.5nm, the emission slit width is 2.5nm, and the integration time is 0.1s; the excitation wavelength of the fluorescence spectrometer is 340nm, the emission wavelength range is 345-650nm, and the step size is 1nm.

[0030] In one embodiment of the present invention, in step (3), the quantitative detection model is: Y=79.41C+10606.47, wherein C represents the concentration of the added L-cysteine ​​solution.

[0031] In one embodiment of the present invention, the method further comprises: processing the serum sample to be tested according to steps (1)-(2) and obtaining the fluorescence recovery degree of the serum sample to be tested; and then obtaining the concentration of L-cysteine ​​in the sample according to the quantitative detection model in step (3).

[0032] In one embodiment of the present invention, the method further comprises:

[0033] (a) Configuration of different Fe 3+ ion concentration of trivalent iron salt solution, the trivalent iron salt solution and the carbon dot solution are mixed evenly to obtain a sample solution, and fluorescence spectrum detection is performed after incubation for a period of time;

[0034] (b) The fluorescence quenching degree and Fe 3+ Linear model for ion concentrations.

[0035] In one embodiment of the present invention, the conditions for fluorescence spectrum detection in step (a) are: the fluorescence spectrum is measured using a fluorescence spectrometer, the excitation slit width of the spectrometer is 2.5nm, the emission slit width is 2.5nm, and the integration time is 0.1s; the excitation wavelength of the fluorescence spectrometer is 340nm, the emission wavelength range is 345-650nm, and the step size is 1nm.

[0036] In one embodiment of the present invention, the linear model described in step (b) is: Y = -206.556C + 50924.634, where C represents the added Fe 3+ concentration.

[0037] In one embodiment of the present invention, Fe 3+ The ion concentration is 0~200μM.

[0038] The invention also provides application of the detection method in manufacturing biological detection equipment.

[0039] Beneficial effects:

[0040] 1. The present invention detects L-cysteine ​​in a milk environment based on N and S co-doped carbon nanodots. The carbon dots are spherical or quasi-spherical in appearance and have rich functional groups on the surface.

[0041] 2. The present invention is the first to synthesize carbon dots using β-mercaptoethylamine and p-phenylenediamine as precursors and use them as fluorescent sensors to achieve quantitative detection of L-cysteine ​​in serum samples. This method is simple, fast, safe, and suitable for routine analysis.

[0042] 3. In the method of the present invention, carbonyl functional groups appear on the surface of N and S co-doped carbon nanodots, which can better combine with Fe3+, thereby having better selectivity for L-cysteine ​​in the subsequent reaction.

[0043] The linear range of the method for detecting L-cysteine ​​is 0-300 μM, and the detection limit is as low as 1.47 μM, which is of great significance in the field of biological detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the detection of L-cysteine ​​in serum based on N and S co-doped carbon nanodots.

[0045] Figure 2 Different concentrations of Fe are added to the system in Example 2. 3+ Fluorescence spectrum of ionic solution.

[0046] Figure 3 The fluorescence quenching degree and Fe in Example 2 3+ The relationship curve of ion concentration.

[0047] Figure 4 The fluorescence quenching degree and concentration range of Fe in Example 2 are 0-200 μM. 3+ Linear fitting curve of ions.

[0048] Figure 5 The carbon dot-Fe in Example 3 3+ Fluorescence spectra when different concentrations of L-cysteine ​​were added to the mixed system.

[0049] Figure 6 This is the relationship curve between the fluorescence recovery degree and the L-cysteine ​​concentration in Example 3.

[0050] Figure 7 It is a linear fitting curve of the fluorescence recovery degree in Example 3 and the L-cysteine ​​concentration range of 0 to 300 μM.

[0051] Figure 8 This is a graph showing the test results for detecting the selectivity of L-cysteine ​​in Example 4. DETAILED DESCRIPTION

[0052] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0053] Example 1 Preparation of N, S co-doped carbon dots (N, S-CDs) solution

[0054] Weigh 0.0771g of β-mercaptoethylamine and 0.1081g of p-phenylenediamine in a beaker, add 25mL of deionized water, and ultrasonically treat for 10min. After the mixture is fully dissolved, move it into a 50mL polytetrafluoroethylene liner and react at 180°C for 8h. The synthesized crude product is purified by a chromatography column. The eluent used during purification is methanol and ethyl acetate, and the ratio is 1:10. The silicon powder used is 300-400 mesh. The solution with the green luminescent part is extracted, and then the solution is concentrated. 3ml of anhydrous ethanol is used to dissolve the carbon dots remaining on the spherical bottle, and then diluted 100 times with an aqueous solution to obtain a carbon dot solution.

[0055] Example 2 Construction of Fe 3+ Linear determination model of ions

[0056] (1) Prepare sample solutions: carbon dot solution (the carbon dot solution diluted 100 times in Example 1), Fe with concentrations of 0 μM (blank control), 5 μM, 10 μM, 20 μM, 50 μM, 70 μM, 100 μM, 150 μM, 200 μM, 350 μM, 500 μM, and 1000 μM, respectively. 3+ Ions (FeCl 3 ) aqueous solution;

[0057] (2) 1 mL of 100-fold diluted carbon dot solution and 1 mL of Fe 3+ The ion-water solution was mixed, the volume was adjusted to 4 mL with deionized water, and incubated for 1 h to obtain different concentrations of Fe 3+ ions were spiked with carbon dot solution and fluorescence spectrometry was performed, and the reaction temperature was 20 °C;

[0058] (3) Fluorescence spectrum of the measurement system: Scanning conditions: excitation wavelength is 340 nm, emission wavelength scanning range is 345-650 nm, scanning is performed every 1 nm, slit width is set to 2.5 nm / 2.5 nm (excitation slit / emission slit), and the obtained fluorescence emission spectrum ( Figure 2 ).

[0059] (4) Plot the fluorescence quenching degree of the sample solution and Fe 3+ The relationship curve of ion concentration, such as Figure 3 As shown. 3+ When the ion concentration is 0-200 μM, the fluorescence quenching degree and Fe 3+ The fitting curve of ion concentration is as follows: Figure 4 As shown in Figure 2, it can be seen that the degree of fluorescence quenching of the solution is related to Fe 3+ The ion concentration is linearly related, the linear equation is Y = -206.556C + 50924.634, and the correlation coefficient is R 2 =0.98319.

[0060] Example 3 Construction of a linear determination model for L-cysteine

[0061] (1) Preparation of sample solutions: carbon dot solution (carbon dot solution diluted 100 times in Example 1), L-cysteine ​​aqueous solutions with concentrations of 0 μM (blank control), 25 μM, 50 μM, 75 μM, 100 μM, 125 μM, 175 μM, 200 μM, 300 μM, 500 μM, 1000 μM, and 1500 μM, respectively.

[0062] (2) 1 mL of carbon dot solution and 1 mL of 200 μM Fe 3+ The ion solution was mixed, and then 1 mL of L-cysteine ​​solution of different concentrations was added and mixed, and the volume was made up to 4 mL with deionized water, and incubated for 1 h to obtain the spiked carbon dot Fe with different concentrations of L-cysteine. 3+ The solution was tested by fluorescence spectroscopy, and the reaction temperature was 20°C;

[0063] (3) Fluorescence spectrum of the measurement system: Scanning conditions: excitation wavelength is 340 nm, emission wavelength scanning range is 345-650 nm, scanning is performed every 1 nm, slit width is set to 2.5 nm / 2.5 nm (excitation slit / emission slit), and the obtained fluorescence emission spectrum ( Figure 5 )

[0064] (4) Draw a curve showing the relationship between the fluorescence recovery degree of the sample solution and the L-cysteine ​​concentration, such as Figure 6 When the concentration of L-cysteine ​​is 0-300 μM, the fitting curve of the fluorescence recovery degree and the concentration of L-cysteine ​​is shown in Figure 7 As shown, it can be seen that the degree of fluorescence recovery of the solution is linearly related to the concentration of L-cysteine, the linear equation is Y=79.41C+10606.47, and the correlation coefficient is R 2 =0.9991, and the detection limit is 1.47 μM.

[0065] Example 4 Selectivity of N and S co-doped carbon dots for L-cysteine

[0066] Referring to Example 3, different types of amino acids (glutathione, histidine, lysine, arginine, tryptophan, alanine, 4-aminobutyric acid, aspartic acid, methionine, glutamine, phenylalanine, asparagine, tyrosine, threonine, proline, glycine, valine, serine, leucine) with a concentration of 500 μM were respectively reacted with carbon dot-Fe 3+ Mix the system solution and deionized water, and then perform fluorescence spectrum detection. Figure 8All fluorescence detections shown were performed under the same conditions. According to the test results, the fluorescence response intensity of the carbon dots to other types of amino acids is relatively low, and they are only selective for L-cysteine. It can be seen that the detection method of the present invention can specifically detect L-cysteine ​​without interference from other amino acids.

[0067] Example 5 Detection of L-cysteine ​​in serum sample environment

[0068] Pre-treat the serum samples: thaw the serum samples naturally at room temperature, vortex them after they are completely thawed to mix the serum sample solution evenly, then take 1 mL of the serum sample and add 49 mL of deionized water to dilute the sample 50 times.

[0069] Referring to Example 3, serum pretreatment solutions containing L-cysteine ​​at concentrations of 25, 50, 75, and 100 were measured. The test results are shown in Table 1.

[0070] Table 1 Test results of Example 5

[0071] Spike concentration (μM) Detection concentration (μM) Recovery rate (%) Relative standard deviation (%), n = 3 25 26.01 104 0.915 50 58.74 117 0.678 75 79.6 106 0.617 100 91.25 91 0.397

[0072] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for detecting L-cysteine ​​in serum based on N and S co-doped carbon nanodots, comprising the following steps: (1) Using β-mercaptoethylamine and p-phenylenediamine as precursors, carbon dots were obtained through hydrothermal reaction; (2) The obtained carbon dots are first mixed with a trivalent iron salt solution to form a carbon dot-Fe 3+ A mixed system solution is then added with a series of L-cysteine ​​solutions of known concentrations, and the mixture is incubated for a period of time. The fluorescence intensity before and after the addition of L-cysteine ​​is then detected by fluorescence spectroscopy to obtain the corresponding fluorescence intensity change value, i.e., the fluorescence recovery degree; (3) Using the fluorescence recovery degree and Fe 3+ The quantitative detection model was constructed based on ion concentration.

2. The method according to claim 1, characterized in that The process of synthesizing carbon dots in step (1) includes: mixing β-mercaptoethylamine, p-phenylenediamine and deionized water, and performing a hydrothermal reaction after fully dissolving; after the reaction is completed, purifying by a chromatography column, then concentrating, and diluting with an aqueous solution to obtain a carbon dot solution.

3. The method according to claim 1, characterized in that In step (1), the mass ratio of β-mercaptoethylamine to p-phenylenediamine is 1:(1.0-2.0); the temperature of the hydrothermal reaction is 150-200° C., and the time is 5-15 hours; and the dilution multiple of the aqueous solution is 100-400 times.

4. The method according to claim 1, characterized in that: In step (1), the eluents used for purification by the chromatography column are methanol and ethyl acetate, and the solution emitting green light is extracted; Step (1) also includes: concentrating the purified solution, dissolving the carbon dots remaining on the spherical bottle with anhydrous ethanol, and finally diluting with an aqueous solution to obtain a carbon dot solution.

5. The method according to claim 1, characterized in that In step (2), the concentration of the ferric salt solution is 100-500 μM; the ferric salt is ferric chloride; and the volume ratio of the carbon dot solution to the ferric salt solution is 1:

1.

6. The method according to claim 1, characterized in that In step (2), L-cysteine ​​solution and carbon dot-Fe 3+ The volume ratio of the mixed system solution is 1:2; the concentration of the L-cysteine ​​solution is 0-300 μM.

7. The method according to claim 1, characterized in that In step (2), the conditions for fluorescence spectrum detection are: the fluorescence spectrum is measured using a fluorescence spectrometer, the excitation slit width of the spectrometer is 2.5nm, the emission slit width is 2.5nm, and the integration time is 0.1s; the excitation wavelength of the fluorescence spectrometer is 340nm, the emission wavelength range is 345-650nm, and the step size is 1nm.

8. The method according to any one of claims 1 to 7, characterized in that: The method further comprises: processing the serum sample to be tested according to the process of steps (1)-(2) and obtaining the fluorescence recovery degree of the serum sample to be tested; and then obtaining the concentration of L-cysteine ​​in the sample according to the quantitative detection model in step (3).

9. A method for simultaneous detection of Fe based on N and S co-doped carbon nanodots 3+ The method is characterized in that The method comprises: (a) Configuration of different Fe 3+ ion concentration of trivalent iron salt solution, the trivalent iron salt solution and the carbon dot solution of claim 1 are mixed evenly to obtain a sample solution, and fluorescence spectrum detection is performed after incubation for a period of time; (b) The fluorescence quenching degree and Fe 3+ Linear model for ion concentrations.

10. Use of the method for detecting L-cysteine ​​in serum based on N and S co-doped carbon nanodots according to any one of claims 1 to 8 in manufacturing biological detection equipment.

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