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

Abstract

The application discloses a method for detecting L-cysteine in serum based on N and S co-doped carbon nanodots, and belongs to the field of analysis and detection. 3+ After that, carbon dot fluorescence quenching phenomenon appears, and a carbon dot-Fe 3+ mixed system is obtained. 3+ After L-cysteine is added into the carbon dot-Fe The application first uses beta-mercaptoethylamine and p-phenylenediamine as precursors to prepare carbon dots, realizes quantitative detection of L-cysteine, has low cost, high sensitivity, is suitable for on-site rapid detection, and has high practical value and popularization prospect.

Description

TECHNICAL FIELD

[0001] The application relates to 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. BACKGROUND

[0002] Cysteine (Cys) is the only amino acid containing free sulfhydryl among 20 common amino acids, and is an important amino acid in the human body. Whether normal changes in the level of cysteine have a significant impact on human function. Abnormal cysteine levels can cause many diseases, such as increased risk of cardiovascular and cerebrovascular diseases, endothelial cell damage, vascular occlusion and nerve damage 2. When the concentration of L-cysteine and cystine in urine is too high (usually up to 100 uM), liver cystinuria occurs, which is a genetic disease of amino acid transport. Therefore, the detection of L-cysteine is of great significance for detecting human health.

[0003] At present, the common detection methods of L-cysteine mainly include chromatography, mass spectrometry and electrochemical voltammetry analysis method, etc. Among them, although chromatography and mass spectrometry have high sensitivity and high accuracy, the equipment is expensive and the operation is complex, which is not suitable for on-site detection, such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) and the like. While electrochemical voltammetry is relatively simple, but it faces the problem of insufficient sensitivity, especially in the detection of low concentration of the measured substance.

[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 existing technology “Application research of fluorescent “off-on” strategy in detection of small thiol molecules” reports a detection system using carbon dots combined with quencher silver nanoparticles (AgNPs), but when detecting L-cysteine by relying on this detection system, it also has a significant response to glutathione (GSH), homocysteine (Hcy) and the like, and cannot realize specific detection of L-cysteine.

[0005] Therefore, it is urgent to develop a simple, efficient, sensitive, wide detection range and specific method for detecting L-cysteine. SUMMARY

[0006] Technical problem:

[0007] In the existing L-cysteine detection technology, although chromatography and mass spectrometry have high accuracy, they have the problems of expensive equipment, complex operation and unsuitability for on-site rapid detection. While electrochemical voltammetry is relatively simple, but it has insufficient sensitivity, especially in the detection of low concentration of L-cysteine, and the effect is not good, which is difficult to meet the efficient and low-cost detection demand. And the existing carbon dot fluorescence detection has a limited detection range and cannot specifically detect L-cysteine.

[0008] Technical solution:

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

[0010] (1) obtaining carbon dots by hydrothermal reaction with β-mercaptoethylamine and p-phenylenediamine as precursors;

[0011] (2) mixing the obtained carbon dots with a ferric salt solution to form a carbon dot-Fe 3+ mixed system solution; then adding a series of L-cysteine solutions with known concentrations, incubating for a period of time, and then detecting the fluorescence intensity before and after the addition of L-cysteine by fluorescence spectroscopy to obtain the corresponding fluorescence intensity change value, i.e. the fluorescence recovery degree;

[0012] (3) constructing a quantitative detection model by using the fluorescence recovery degree and the Fe 3+ ion concentration in the ferric salt solution.

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

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

[0015] In an embodiment of the application, the temperature of the hydrothermal reaction is 150-200℃. Specifically, 180℃ can be selected.

[0016] In an embodiment of the application, the time of the hydrothermal reaction is 5-15h. Specifically, 8h can be selected.

[0017] In an embodiment of the application, the dilution multiple of the aqueous solution is 100-400 times.

[0018] In an embodiment of the application, the process of synthesizing carbon dots in step (1) specifically comprises: weighing 0.0771g of β-mercaptoethylamine and 0.1081g of p-phenylenediamine in a beaker, adding 25mL of deionized water, ultrasonic treating for 10min, fully dissolving the mixture and then moving it into a 50mL polytetrafluoroethylene liner, and reacting at 180℃ for 8h.

[0019] In an embodiment of the application, when the chromatographic column is used for purification, the eluent used is methanol and ethyl acetate, and the solution with green light emission is extracted.

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

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

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

[0023] In one embodiment of the present application, in step (2), the concentration of the ferric salt solution is 100-500 μM. Specifically, 200 μM can be selected.

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

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

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

[0027] In one embodiment of the present application, in step (2), the incubation time is 0.5-2 h. Specifically, 1 h can be selected.

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

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

[0030] In one embodiment of the present application, 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 application, 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; then obtaining the concentration of L-cysteine in the sample according to the quantitative detection model in step (3).

[0032] In an embodiment of the present application, the method further comprises:

[0033] (a) configuring different Fe 3+ ion concentrations of the ferric salt solution, mixing the ferric salt solution and the carbon dot solution uniformly to obtain a sample solution, and performing fluorescence spectrum detection after incubation for a period of time;

[0034] (b) constructing a linear model of fluorescence quenching degree and Fe 3+ ion concentration according to the change of fluorescence intensity before and after adding the ferric salt solution.

[0035] In an embodiment of the present application, the fluorescence spectrum detection in step (a) is performed under the following conditions: the fluorescence spectrum is measured by a fluorescence spectrometer, the excitation slit width of the spectrometer is 2.5 nm, the emission slit width is 2.5 nm, and the integration time is 0.1 s; the excitation wavelength of the fluorescence spectrometer is 340 nm, the emission wavelength range is 345-650 nm, and the step is 1 nm.

[0036] In an embodiment of the present application, the linear model in step (b) is Y = -206.556C + 50924.634, wherein C represents the concentration of Fe 3+ .

[0037] In an embodiment of the present application, the Fe 3+ ion concentration in the ferric salt solution is 0-200 μM.

[0038] The present application also provides the use of the detection method in the manufacture of biological detection equipment.

[0039] Advantages:

[0040] 1. The present application detects L-cysteine in milk environment based on N, S co-doped carbon nanodots, and the carbon dots are spherical or spherical-like in appearance and contain abundant functional groups on the surface.

[0041] 2. The present application first synthesizes carbon dots using β-mercaptoethylamine and p-phenylenediamine as precursors, and realizes quantitative detection of L-cysteine in serum samples as a fluorescence sensor. This method is simple, fast, safe and suitable for routine analysis.

[0042] 3. In the method of the present application, the N, S co-doped carbon nanodots have carbonyl functional groups on the surface, which can better bind with Fe3+, thereby having good selectivity for L-cysteine subsequently.

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

[0044] Figure 1 The schematic diagram for detecting L-cysteine in serum based on N, S co-doped carbon nanodots.

[0045] Figure 2 The fluorescence spectra of the system in Example 2 with different concentrations of Fe 3+ ion solution.

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

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

[0048] Figure 5 The fluorescence spectra of the carbon dot-Fe 3+ mixed system in Example 3 with different concentrations of L-cysteine.

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

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

[0051] Figure 8 The test result diagram of the selectivity for detecting L-cysteine in Example 4. DETAILED DESCRIPTION

[0052] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application.

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

[0054] Take 0.0771 g of β-mercaptoethylamine and 0.1081 g of p-phenylenediamine in a beaker, add 25 mL of deionized water, ultrasonic treatment for 10 min, after the mixture is fully dissolved, move into a 50 mL polytetrafluoroethylene liner, react at 180℃ for 8h. The crude product of synthesis is purified by the method of chromatographic column, the eluent used in purification is methanol and ethyl acetate, the ratio is 1:10, the silica powder used is 300-400 mesh, the solution of the part with green light emission is extracted, then the solution is concentrated, 3ml of anhydrous ethanol is used to dissolve the residue on the carbon dot ball bottle, then the solution is diluted 100 times with aqueous solution to obtain the carbon dot solution.

[0055] Example 2 Construction of Fe 3+ Linear Determination Model of Ions

[0056] (1) Preparation of sample solution: carbon dot solution (carbon dot solution diluted 100 times in Example 1), Fe 3+ ion (FeCl3) aqueous solution 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, 1000 μM, respectively;

[0057] (2) Mix 1 mL of carbon dot solution diluted 100 times and 1 mL of Fe 3+ ion aqueous solution with different concentrations, make up to 4 mL with deionized water, incubate for 1 h to obtain carbon dot solution with different concentrations of Fe 3+ ions, and detect the fluorescence spectrum at a reaction temperature of 20℃;

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

[0059] (4) Draw the relationship curve between the fluorescence quenching degree of the sample solution and the concentration of Fe 3+ ions, as shown in Figure 3 . When the concentration of Fe 3+ ions is 0-200 μM, the fitting curve of the fluorescence quenching degree and the concentration of Fe 3+ ions is as shown in Figure 4 . It can be seen that the fluorescence quenching degree of the solution has a linear relationship with the concentration of Fe 3+ ions, the linear equation is Y=-206.556C+50924.634, and the correlation coefficient is R 2 =0.98319.

[0060] Example 3 Constructing a linear assay model for L-cysteine

[0061] (1) Preparation of sample solution: carbon dot solution (carbon dot solution diluted 100 times in Example 1), L-cysteine aqueous solution 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, 1500 μM, respectively

[0062] (2) Mix 1 mL of carbon dot solution and 1 mL of 200 μM Fe 3+ ion solution, then add 1 mL of L-cysteine solution with different concentrations, and mix with deionized water to a volume of 4 mL, and incubate for 1 h to obtain a carbon dot Fe 3+ solution with different concentrations of L-cysteine, and perform fluorescence spectrum detection, with a reaction temperature of 20°C;

[0063] (3) Measurement of fluorescence spectrum of the system: scanning conditions: excitation wavelength of 340 nm, emission wavelength scanning range of 345-650 nm, scanning every 1 nm, slit width of 2.5 nm / 2.5 nm (excitation slit / emission slit), and the obtained fluorescence emission spectrum is as shown in Figure 5 )

[0064] (4) Draw the relationship curve between the fluorescence recovery degree of the sample solution and the concentration of L-cysteine, as shown in 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 as shown in Figure 7 It can be seen that the fluorescence recovery degree of the solution has a linear relationship with the concentration of L-cysteine, the linear equation is Y = 79.41C + 10606.47, the correlation coefficient is R 2 = 0.9991, and the detection limit is 1.47 μM.

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

[0066] Referring to Example 3, different kinds 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 mixed with the carbon dot-Fe 3+ mixed system solution and deionized water, and then fluorescence spectrum detection was performed. As Figure 8All the fluorescence detection shown is carried out under the same condition. According to the detection result, it can be known that the fluorescence response intensity of the carbon dots to other kinds of amino acids is relatively low, and only has selectivity to L-cysteine. It can be seen that the detection method of the present application can specifically detect L-cysteine and is not interfered by other amino acids.

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

[0068] The serum sample is pretreated: the serum sample is naturally thawed at room temperature, vortexed after complete thawing, and then 1 mL of the serum sample is taken and diluted with 49 mL of deionized water to make the sample 50 times diluted

[0069] Referring to Example 3, the serum pretreatment solution containing L-cysteine with a concentration of 25, 50, 75 and 100 is measured, and the detection result is shown in Table 1.

[0070] Table 1 Test result of Example 5

[0071] Spiked concentration (pM) Detection concentration (pM) Recovery (%) 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 above provided examples are not used to limit the scope covered by the present application, and the described steps are not used to limit the execution order. The person skilled in the art makes obvious improvements to the present application combined with the existing common knowledge, which also falls within the protection scope defined by the claims of the present application.

Claims

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

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

3. The method of claim 2, wherein, 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℃, and the time is 5-15h; the dilution multiple of the aqueous solution is 100-400 times.

4. The method of claim 2, wherein, In step (1), the eluent used when purifying through a chromatographic column is methanol and ethyl acetate, and the solution with green light emission is extracted; In step (1), it further comprises: concentrating the solution obtained after purification, dissolving the carbon dots remaining on the spherical bottle with anhydrous ethanol after concentration, and finally diluting with an aqueous solution to obtain a carbon dot solution.

5. The method of claim 1, wherein, In step (2), the concentration of the ferric salt solution is 100-500μM; 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 of claim 1, wherein, In step (2), the L-cysteine solution and the carbon dots-Fe 3+ The volume ratio of the mixed system solution was 1:2, and the concentration of the L-cysteine solution was 0-300 μM.

7. The method of claim 1, wherein, In step (2), the fluorescence spectrum detection conditions are: using a fluorescence spectrometer to measure the fluorescence spectrum, 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 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 processes of steps (1)-(2) and obtaining the fluorescence recovery degree of the serum sample to be tested; then obtaining the concentration of L-cysteine in the sample according to the quantitative detection model in step (3). 9.Use of the method for detecting L-cysteine in serum based on N, S co-doped carbon nanodots according to any one of claims 1-8 in the manufacture of a biological detection device.

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