Application of nano CDs as fluorescent probe in fluorescence detection of sugammadex sodium and fluorescence detection method of sugammadex sodium

Abstract

The invention provides application of nano CDs as a fluorescent probe in fluorescence detection of sugammadex sodium and a fluorescence detection method of sugammadex sodium, and relates to the technical field of drug analysis and detection. According to the invention, carboxyl in sugammadex sodium and nitrogen atoms in nano CDs interact with each other, the fluorescence intensity of nano CDs is enhanced through intramolecular charge transfer, sugammadex sodium and nano CDs can be effectively combined through a static combination effect, and nano CDs have high sensitivity and selectivity to sugammadex sodium; the method is suitable for detecting sugammadex sodium. By adopting a fluorescence analysis method, the high-sensitivity, high-selectivity, simple, rapid and accurate quantitative detection of the sugammadex can be realized according to the fluorescence enhancement efficiency of the sugammadex relative to the nano CDs after the sugammadex is added and a standard curve of the concentration of the sugammadex and the fluorescence enhancement efficiency. The fluorescence detection method provided by the invention is low in detection cost.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis and detection, and in particular to an application of nano-CDs as a fluorescent probe in the fluorescence detection of sugammadex sodium and a fluorescence detection method of sugammadex sodium. Background Art

[0002] Sugammadex sodium (SUG) is a derivative of γ-cyclodextrin and can be used as a clinical intraoperative muscle relaxant antagonist. It can be selectively combined with rocuronium or vecuronium to reverse neuromuscular blockade. However, a variety of postoperative complications, including impaired pharyngeal function, decreased functional residual capacity, and impaired hypoxic ventilatory response, are associated with the residue of SUG. Currently, the most commonly used method for the analysis and detection of SUG is high performance liquid chromatography (HPLC). For example, there are methods that use organosilicon polymer coated silica particles bonded to adamantane chromatographic columns, ghost peak capture columns connected in series with C 18 There are reports on high performance liquid chromatography detection methods using chromatographic columns or charged aerosol detectors (CAD). However, the above methods are complicated and time-consuming. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide an application of nano-CDs as a fluorescent probe in the fluorescence detection of sugammadex sodium and a fluorescence detection method of sugammadex sodium. The present invention uses nano-CDs to detect sugammadex sodium, which is simple, rapid and highly sensitive.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides application of nano CDs as fluorescent probes in fluorescence detection of sugammadex sodium, wherein the nano CDs contain nitrogen atoms.

[0005] Preferably, the particle size of the nano-CDs is less than 5 nm.

[0006] Preferably, the raw materials for preparing the nano-CDs include polyethyleneimine and bromophenol blue.

[0007] Preferably, the method for preparing the nano-CDs comprises the following steps: mixing polyethyleneimine, bromophenol blue and water, and performing a hydrothermal reaction to obtain the nano-CDs.

[0008] The present invention also provides a fluorescence detection method for sugammadex sodium, comprising the following steps: The probe mixture of the sample to be tested and the probe solution are respectively subjected to fluorescence detection to obtain the fluorescence intensity to be tested and the initial fluorescence intensity; the probe mixture of the sample to be tested comprises a sugammadex sodium sample to be tested, nano-CDs and an aqueous phase, and the probe solution is different from the probe mixture of the sample to be tested in that it does not contain the sugammadex sodium sample to be tested; Obtaining the fluorescence enhancement efficiency of the sugammadex sodium sample to be tested according to the ratio of the fluorescence intensity to be tested to the initial fluorescence intensity; The quantitative detection result of sugammadex sodium is obtained according to the fluorescence enhancement efficiency of the sugammadex sodium sample to be tested and the standard curve; the standard curve is a standard curve with the concentration of sugammadex sodium as the independent variable and the fluorescence enhancement efficiency as the dependent variable.

[0009] Preferably, the test conditions of the fluorescence detection include: the fluorescence excitation wavelength is 365nm; the fluorescence emission wavelength detection range is 380~540nm; the slit width is 5nm; the fluorescence intensity at 460nm is the fluorescence intensity to be measured or the initial fluorescence intensity.

[0010] Preferably, the linear range of the standard curve is 0.001-0.07 or 0.07-0.5 g / L.

[0011] Preferably, the aqueous phase comprises water or a phosphate buffer solution.

[0012] Preferably, the pH value of the phosphate buffer solution is 4-10.

[0013] Preferably, the phosphate in the phosphate buffer solution includes at least one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, and diammonium hydrogen phosphate; The total concentration of phosphate in the phosphate buffer solution is 0.01-0.1 mol / L.

[0014] In the present invention, the carboxyl groups in sodium sugammadex and the nitrogen atoms in nano-CDs interact with each other, and the fluorescence intensity is enhanced by intramolecular charge transfer, so that sodium sugammadex has the characteristic of enhancing the fluorescence of nano-CDs, and sodium sugammadex and nano-CDs can be effectively combined through static binding. At the same time, nano-CDs have high sensitivity and selectivity to sodium sugammadex, and nano-CDs are suitable for the detection of sodium sugammadex. The present invention adopts fluorescence analysis, according to the fluorescence enhancement efficiency relative to nano-CDs after adding sodium sugammadex, and the standard curve with the concentration of sodium sugammadex as the independent variable and the fluorescence enhancement efficiency as the dependent variable, the highly sensitive, highly selective, simple, rapid and accurate quantitative detection of sodium sugammadex can be achieved. And the fluorescence detection method provided by the present invention has low detection cost.

[0015] The fluorescence detection method provided by the present invention is applicable to acidic, neutral and alkaline conditions. Sugammadex sodium shows fluorescence enhancement on nano-CDs, and the fluorescence enhancement efficiency remains basically unchanged. The fluorescence detection method provided by the present invention is applicable to a wide pH range. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1is a transmission electron microscopy image of the nano-CDs prepared in Example 1; Figure 2 This is the fluorescence emission spectrum of sugammadex sodium in response to nano-CDs in Example 2; Figure 3 The fluorescence decay spectrum and fluorescence lifetime diagram of sugammadex sodium on nano-CDs in Example 3; Figure 4 The Zeta potential diagram of the nano-CDs in Example 4 before and after the addition of sugammadex sodium; Figure 5 The fluorescence emission spectra of the nano-CDs titrated with different sugammadex sodium concentrations in Example 5; Figure 6 The linear standard curve of fluorescence enhancement efficiency-sugammadex sodium concentration in Example 6; Figure 7 This is the selectivity spectrum of the nano-CDs for different sugammadex sodium detection in Example 7; Figure 8 The fluorescence response spectrum of the functional groups and structures of sodium sugammadex to nano-CDs in Example 8; Fig. 9 The fluorescence enhancement efficiency of sodium sugammadex on nano-CDs in different pH solutions in Example 9 is shown in FIG. Fig.10 This is a graph showing the fluorescence enhancement efficiency of sugammadex sodium on nano-CDs at different reaction times in Example 10; Fig.11 This is the fluorescence enhancement spectrum of the clinically used sodium sugammadex injection on nano-CDs in Example 11; Fig.12 This is the fluorescence emission spectrum of sugammadex sodium in response to nano-CDs in Example 12; Fig.13 This is the fluorescence emission spectrum of sugammadex sodium in response to nano-CDs in Example 13; Fig.14 This is the fluorescence emission spectrum of sugammadex sodium in Comparative Example 1 in response to the CDs fluorescent probe that does not contain nitrogen atoms. DETAILED DESCRIPTION

[0017] The invention provides application of nano CDs as fluorescent probes in fluorescence detection of sugammadex sodium, wherein the nano CDs contain nitrogen atoms.

[0018] In the present invention, the particle size of the nano-CDs is preferably less than 5 nm.

[0019] In the present invention, the carboxyl groups in sugammadex sodium and the nitrogen atoms in nano-CDs interact with each other, and the fluorescence intensity is enhanced through intramolecular charge transfer. Meanwhile, nano-CDs have high sensitivity and selectivity to sugammadex sodium, and nano-CDs are suitable for the detection of sugammadex sodium.

[0020] In the present invention, the method for preparing the nano-CDs preferably comprises the following steps: mixing polyethyleneimine, bromophenol blue and water, and performing a hydrothermal reaction to obtain the nano-CDs.

[0021] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0022] In the present invention, the mass ratio of polyethyleneimine to bromophenol blue is preferably 1:0.01-0.2, and in specific embodiments may be 1:0.01, 1:0.05, 1:0.1, 1:0.15 or 1:0.2.

[0023] In the present invention, the ratio of the mass of the polyethyleneimine to the volume of water is preferably 1 g:100-200 mL, and in specific embodiments may be 1 g:100 mL, 1 g:150 mL or 1 g:200 mL.

[0024] In the present invention, the temperature of the hydrothermal reaction is preferably 150-200°C, and in specific embodiments it can be 150°C, 160°C, 170°C, 180°C, 190°C or 200°C; the time of the hydrothermal reaction is preferably 6-18h, and in specific embodiments it can be 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h or 18h.

[0025] After the hydrothermal reaction is completed, the present invention further comprises: performing water dialysis on the hydrothermal reaction liquid obtained by the hydrothermal reaction to obtain a nano-CDs mother liquor, diluting the nano-CDs mother liquor with water to obtain a nano-CDs solution, and the nano-CDs solution is directly used for subsequent fluorescence detection. In the present invention, the molecular weight cutoff of the dialysis bag used for the water dialysis is preferably 3000-5000Da, and in a specific embodiment, it can be 3000Da, 3500Da, 4000Da, 4500Da or 5000Da; the temperature of the water dialysis is preferably room temperature, and the time of the water dialysis is preferably 12-24h, and in a specific embodiment, it can be 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h or 24h; the water used for the water dialysis preferably includes secondary water. In the present invention, the concentration of the nano CDs solution is preferably 0.056-0.56 mg / mL, and in specific embodiments can be 0.056 mg / mL, 0.11 mg / mL, 0.17 mg / mL, 0.22 mg / mL, 0.28 mg / mL, 0.33 mg / mL, 0.39 mg / mL, 0.34 mg / mL, 0.50 mg / mL, 0.56 mg / mL. In the present invention, the aqueous phase preferably includes water or a phosphate buffer solution; the parameters of the phosphate buffer solution are described in the fluorescence detection method.

[0026] In the present invention, the detection preferably comprises fluorescence detection.

[0027] In the present invention, sugammadex sodium has the property of enhancing the fluorescence of CDs, and sugammadex sodium can be effectively combined with nano-CDs through static binding. At the same time, nano-CDs have a high sensitivity to sugammadex sodium. Therefore, nano-CDs have the ability to quantitatively analyze sugammadex sodium and can be used for fluorescence detection of sugammadex sodium.

[0028] The present invention also provides a fluorescence detection method for sugammadex sodium, comprising the following steps: The probe mixture of the sample to be tested and the probe solution are respectively subjected to fluorescence detection to obtain the fluorescence intensity to be tested and the initial fluorescence intensity; the probe mixture of the sample to be tested comprises a sugammadex sodium sample to be tested, nano-CDs and an aqueous phase, and the probe solution is different from the probe mixture of the sample to be tested in that it does not contain the sugammadex sodium sample to be tested; Obtaining the fluorescence enhancement efficiency of the sugammadex sodium sample to be tested according to the ratio of the fluorescence intensity to be tested to the initial fluorescence intensity; The quantitative detection result of sugammadex sodium is obtained according to the fluorescence enhancement efficiency of the sugammadex sodium sample to be tested and the standard curve; the standard curve is a standard curve with the concentration of sugammadex sodium as the independent variable and the fluorescence enhancement efficiency as the dependent variable.

[0029] The present invention performs fluorescence detection on a sample probe mixture and a probe solution to be tested respectively to obtain a fluorescence intensity to be tested and an initial fluorescence intensity; the sample probe mixture comprises a sugammadex sodium sample to be tested, nano-CDs and an aqueous phase, and the probe solution differs from the sample probe mixture in that it does not contain the sugammadex sodium sample to be tested.

[0030] In the present invention, the aqueous phase preferably includes water or a phosphate buffer solution. In the present invention, the phosphate buffer solution preferably includes phosphate and water; the phosphate preferably includes at least one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, and diammonium hydrogen phosphate; the total concentration of phosphate in the phosphate buffer solution is preferably 0.01-0.1 mol / L, and in specific embodiments, it can be 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L or 0.1 mol / L. In the present invention, the phosphate buffer solution preferably further comprises a pH regulator, the pH regulator preferably comprises an acid and / or a base, the acid preferably comprises hydrochloric acid and / or sulfuric acid, and the base preferably comprises sodium hydroxide and / or potassium hydroxide; the pH value of the phosphate buffer solution is preferably 4 to 10, and in specific embodiments may be 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 8, 8.5, 9, 9.5 or 10. In the fluorescence detection method provided by the present invention, in a phosphate buffer solution with a pH of 4 to 10, sodium sugammadex exhibits fluorescence enhancement on nano-CDs, and the fluorescence enhancement efficiency remains substantially unchanged, and the fluorescence detection method provided by the present invention is applicable to a wide pH range.

[0031] In the present invention, the nano-CDs are preferably used in the form of a nano-CDs solution, more preferably in the form of a nano-CDs solution obtained by the aforementioned preparation method.

[0032] In the present invention, the preparation method of the sample probe mixture to be tested comprises the following steps: mixing the sample of sodium gluconate to be tested, nano-CDs and an aqueous phase to obtain the sample probe mixture to be tested. In the present invention, the mixing preferably comprises: dissolving the sample of sodium gluconate to be tested in an aqueous phase to obtain a sodium gluconate sample solution, and mixing the sodium gluconate sample solution with a nano-CDs solution; the volume ratio of the sodium gluconate sample solution to the nano-CDs solution is preferably 1:1 to 9, and in a specific embodiment, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 or 1:9. In the present invention, the mixing temperature is preferably room temperature, and the mixing time of the sodium gluconate sample solution and the nano-CDs solution is preferably 5 to 30 minutes, and in a specific embodiment, it can be 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes or 30 minutes.

[0033] In the present invention, the conditions for fluorescence detection preferably include: fluorescence excitation wavelength of 365nm; fluorescence emission wavelength detection range of 380-540nm; slit width of 5nm; fluorescence intensity at 460nm is the fluorescence intensity to be measured or the initial fluorescence intensity.

[0034] In the present invention, the concentration of nano-CDs in the probe solution is the same as that in the sample probe mixture to be tested; the concentration of nano-CDs in the sample probe mixture to be tested is preferably 0.028-0.5 mg / mL, and in specific embodiments it can be 0.028 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.15 mg / mL, 0.17 mg / mL, 0.2 mg / mL, 0.22 mg / mL, 0.25 mg / mL, 0.28 mg / mL, 0.3 mg / mL, 0.33 mg / mL, 0.39 mg / mL, 0.34 mg / mL, 0.4 mg / mL, 0.45 mg / mL or 0.50 mg / mL.

[0035] After obtaining the fluorescence intensity to be measured and the initial fluorescence intensity, the present invention obtains the fluorescence enhancement efficiency of the sugammadex sodium sample to be measured according to the ratio of the fluorescence intensity to be measured and the initial fluorescence intensity, that is, the fluorescence enhancement efficiency=F / F0, F0 is the initial fluorescence intensity, and F is the fluorescence intensity to be measured.

[0036] In the present invention, sodium sugammadex has the property of enhancing the fluorescence of nano-CDs, and sodium sugammadex can be effectively combined with nano-CDs through static binding. At the same time, nano-CDs have high sensitivity and selectivity to sodium sugammadex. The present invention adopts fluorescence analysis method, according to the fluorescence enhancement efficiency relative to nano-CDs after adding sodium sugammadex and the standard curve with the concentration of sodium sugammadex as the independent variable and the fluorescence enhancement efficiency as the dependent variable, it can achieve highly sensitive, highly selective, simple, fast and accurate quantitative detection of sodium sugammadex. And the fluorescence detection method provided by the present invention has low detection cost. The fluorescence detection method provided by the present invention is suitable for acidic, neutral and alkaline conditions. Sodium sugammadex shows fluorescence enhancement to nano-CDs, and the fluorescence enhancement efficiency remains basically unchanged. The fluorescence detection method provided by the present invention is applicable to a wide range of pH.

[0037] After obtaining the fluorescence enhancement efficiency of the sugammadex sodium sample to be tested, the present invention obtains the quantitative detection result of sugammadex sodium according to the fluorescence enhancement efficiency of the sugammadex sodium sample to be tested and a standard curve; the standard curve is a standard curve with the concentration of sugammadex sodium as the independent variable and the fluorescence enhancement efficiency as the dependent variable.

[0038] In the present invention, the method for obtaining the standard curve preferably includes the following steps: preparing a series of sugammadex sodium standard solutions; mixing the series of sugammadex sodium standard solutions with nano-CDs respectively to obtain a series of mixed solutions, and testing the fluorescence intensity of the series of mixed solutions, which is recorded as F; replacing the sugammadex sodium standard solutions in the series of mixed solutions with an equal amount of water to obtain a series of nano-CDs diluted solutions, and testing the fluorescence intensity of the series of nano-CDs diluted solutions, which is recorded as F0; obtaining a series of fluorescence enhancement efficiencies (F / F0) according to the ratio of the fluorescence intensity of the series of mixed solutions to the fluorescence intensity of the series of nano-CDs diluted solutions, drawing a standard curve with the concentration of sugammadex sodium as the independent variable and the fluorescence enhancement efficiency as the dependent variable, and fitting a linear regression equation. In the present invention, the concentrations of the series of sodium gluconate standard solutions are preferably 0.01g / L, 0.03g / L, 0.05g / L, 0.07g / L, 0.1g / L, 0.3g / L, 0.5g / L, 0.7g / L, 1g / L, 3g / L and 5g / L, respectively; the solvent in the series of sodium gluconate standard solutions is preferably water, and the water preferably includes secondary water. In the present invention, the nano-CDs is used in the form of the nano-CDs solution, and the volume ratio of the series of sodium gluconate standard solutions to the nano-CDs solution is preferably 1:9; the solvent in the nano-CDs solution is preferably a phosphate buffer solution of pH=7, and the total concentration of phosphate in the phosphate buffer solution is preferably 0.1mol / L. In the present invention, the conditions of the fluorescence detection are the same as those of the aforementioned fluorescence detection, and will not be repeated here.

[0039] In the present invention, the linear range of the standard curve (i.e., the concentration of sugammadex sodium) is preferably 0.001-0.07 g / L or 0.07-0.5 g / L. In the present invention, when the concentration of sugammadex sodium is 0.001-0.07 g / L, the linear regression equation obtained by fitting the standard curve is preferably Y=1.0538+4.8116X(R 2 =0.9907); when the concentration of the sodium sugammadex is 0.07-0.5 g / L, the linear regression equation obtained by fitting the standard curve is preferably Y=1.3455+0.7941X (R 2 =0.9988). Within the above concentration range, nano-CDs can quantitatively analyze and detect the concentration of sugammadex sodium with a wide detection range and good linear relationship.

[0040] To further illustrate the present invention, the application of the nano-CDs provided by the present invention as a fluorescent probe in the fluorescence detection of sugammadex sodium and the fluorescence detection method of sugammadex sodium are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0041] In the following examples, the detection conditions of fluorescence intensity are as follows: fluorescence excitation wavelength is 365 nm; fluorescence emission wavelength detection range is 380-540 nm; slit width is 5 nm; and fluorescence intensity at 460 nm is recorded. The water used is secondary water.

[0042] Example 1 Dissolve 0.1 g of polyethyleneimine in 20 mL of water and stir until completely dissolved. Add 0.01 g of bromophenol blue powder and dissolve it in the above solution. After stirring, transfer it to a reactor and react at 180°C for 12 h. Cool it to room temperature and transfer it to a 3500 Da dialysis bag for 12 h to obtain a mother solution of nano-CDs.

[0043] The nano-CDs solution mother solution was diluted with water to obtain a nano-CDs aqueous solution with a concentration of 0.28 mg / mL.

[0044] The nano-CDs solution mother solution was diluted with a phosphate buffer solution with a pH of 7 and a total phosphate concentration of 0.mol / L to obtain a nano-CDs phosphate buffer solution with a concentration of 0.28 mg / mL.

[0045] Figure 1 This is a transmission electron microscope image of nano-CDs. It can be seen that the nano-CDs appear as spherical particles with a size less than 5nm, indicating that the probe is a nanodot.

[0046] Example 2 Accurately weigh 0.2178g of sodium sugammadex powder, dissolve it in 10mL of water to obtain 10mmol / L sodium sugammadex mother liquor, and dilute it with water to obtain 0.1mmol / L sodium sugammadex solution. Mix the above sodium sugammadex solution with the nano water CDs solution prepared in Example 1 at a volume ratio of 1:9 at room temperature for 5 minutes to obtain a mixed solution. Replace the sodium sugammadex solution in the mixed solution with an equal amount of water to obtain a nano CDs diluted solution. Test the fluorescence emission spectra of the nano CDs diluted solution and the mixed solution respectively, and the results are shown in Figure 2 In the figure, the horizontal axis is the emission wavelength and the vertical axis is the fluorescence intensity. It can be seen that nano-CDs exhibit fluorescence emission under 365nm excitation, and the emission wavelength is 460nm (CDs, solid line in the figure). After adding sugammadex sodium solution (0.1mmol / L), the fluorescence intensity is significantly enhanced (CDs+SUG, dotted line in the figure), indicating that sugammadex sodium has the characteristic of enhancing the fluorescence of nano-CDs.

[0047] Example 3 Accurately weigh 0.2178g of sodium sugammadex powder, dissolve it in 10mL of water to obtain 10mmol / L sodium sugammadex mother liquor, and dilute it with water to obtain 0.1mmol / L sodium sugammadex solution. Mix the above sodium sugammadex solution with the nano water CDs solution prepared in Example 1 at a volume ratio of 1:5 at room temperature for 5 minutes to obtain a mixed solution. Replace the sodium sugammadex solution in the mixed solution with an equal amount of water to obtain a nano CDs diluted solution. Test the fluorescence attenuation spectra of the nano CDs diluted solution and the mixed solution respectively, and the results are shown in Figure 3 As can be seen from the figure, the fluorescence decay spectra of nano-CDs before and after the addition of sugammadex sodium are almost completely consistent. Through fitting, the fluorescence lifetimes are 5.60ns and 6.12ns, respectively, and the lifetimes are almost unchanged, indicating that there is a static binding interaction between sugammadex sodium and nano-CDs.

[0048] Example 4 Accurately weigh 0.2178g of sodium sugammadex drug powder, dissolve it in 10mL of water to obtain 10mmol / L sodium sugammadex mother liquor, and dilute it with water to obtain 0.1mmol / L sodium sugammadex solution. Mix the above sodium sugammadex solution with the nano water CDs solution prepared in Example 1 at a volume ratio of 1:2 at room temperature for 5 minutes to obtain a mixed solution. Replace the sodium sugammadex solution in the mixed solution with an equal amount of water to obtain a nano CDs diluted solution. Test the Zeta potential of the nano CDs diluted solution and the mixed solution respectively, and the results are shown in Figure 4As can be seen from the figure, the Zeta potential value of nano-CDs decreased from +3.85mV to -11.60mV before and after the addition of sugammadex sodium, indicating that sugammadex sodium and nano-CDs are effectively combined.

[0049] Example 5 0.1000g of sodium sugammadex drug powder was dissolved in 10mL of water to obtain 10g / L sodium sugammadex standard mother solution, which was diluted with water to obtain 0.01g / L, 0.03g / L, 0.05g / L, 0.07g / L, 0.1g / L, 0.3g / L, 0.5g / L, 0.7g / L, 1g / L, 3g / L, and 5g / L sodium sugammadex standard solutions. A series of sodium sugammadex standard solutions were mixed with the nano-water CDs solution prepared in Example 1 at a volume ratio of 1:9 at room temperature for 5 minutes, and the fluorescence emission spectrum of the obtained mixed solution was tested. The results are shown in FIG. Figure 5 As can be seen from the figure, as the concentration of sugammadex sodium increases from 0.001 g / L to 0.5 g / L, the fluorescence intensity of nano-CDs gradually increases, indicating that nano-CDs have a high sensitivity to sugammadex sodium and have the ability to quantitatively analyze sugammadex sodium.

[0050] Example 6 Obtaining the linear regression equation 0.1000g of sodium gluconate raw material powder was dissolved in 10mL of water to obtain 10g / L sodium gluconate standard mother solution, which was diluted with water to obtain 0.01g / L, 0.03g / L, 0.05g / L, 0.07g / L, 0.1g / L, 0.3g / L, 0.5g / L, 0.7g / L, 1g / L, 3g / L, and 5g / L sodium gluconate series standard solutions. The sodium gluconate series standard solutions were mixed with the nano-CDs phosphate buffer solution at a volume ratio of 1:9 at room temperature for 5min to obtain a series of mixed solutions, and the fluorescence intensity of the series of mixed solutions was tested and recorded as F. The sodium gluconate solution in the series of mixed solutions was replaced with an equal amount of water to obtain a series of nano-CDs dilution solutions, and the initial fluorescence intensity of the series of nano-CDs dilution solutions was tested and recorded as F0. The fluorescence enhancement efficiency F / F0 was calculated, and the concentration of sugammadex sodium was used as the independent variable and the fluorescence enhancement efficiency was used as the dependent variable. A linear regression equation was fitted to obtain the linear regression equation for testing sugammadex sodium.

[0051] Figure 6The fluorescence enhancement efficiency-sugammadex sodium concentration linear standard curve is shown in Figure 2. It can be seen that in the range of sugammadex sodium concentration of 0.001~0.07g / L and 0.07~0.5g / L, the fluorescence enhancement efficiency (F / F0) of nano-CDs and the sugammadex sodium concentration show a good linear relationship. When the sugammadex sodium concentration is 0.001~0.07g / L, the linear regression equation is Y=1.0538+4.8116X(R 2 =0.9907), when the concentration of sugammadex sodium is 0.07~0.5g / L, the linear regression equation is Y=1.3455+0.7941X (R 2 =0.9988). This indicates that within the above concentration range, the nanoprobe is able to quantitatively analyze and detect the concentration of sugammadex sodium with a wide detection range and good linear relationship. The detection limit (3σ) was calculated to be 0.44 mg / L, indicating that the nano-CDs has a high sensitivity in detecting sugammadex sodium.

[0052] Example 7 Accurately weigh 0.2178g of sodium sugammadex powder and dissolve it in 10mL of water to obtain 10mmol / L sodium sugammadex mother liquor, and dilute it with water to obtain 0.1mmol / L sodium sugammadex solution. The sodium sugammadex solution was mixed with the nano-CDs solution prepared in Example 1 at a volume ratio of 1:9 at room temperature for 5 minutes to obtain a sodium sugammadex probe mixed solution. The fluorescence emission spectrum of the obtained sodium sugammadex probe mixed solution was tested, and the results are shown in Figure 7 .

[0053] Test of the fluorescence intensity response of other substances to nano-CDs: A single aqueous solution of other substances with a concentration of 0.1 mmol / L was mixed with the nano-CDs phosphate buffer solution prepared in Example 1 at a volume ratio of 1:9 at room temperature for 5 minutes to obtain a mixed solution of other substance probes. The fluorescence emission spectrum of the obtained mixed solution of other substance probes was tested. The results are shown in Figure 7 . Among them, the other substances are: sodium ion, zinc ion, ammonium ion, proline, aspartic acid, threonine, leucine, glycine, lysine, methionine, tryptophan, glutamic acid, arginine, serine, cysteine, alanine, histidine, uric acid, urea, bovine serum albumin, glucose and dopamine.

[0054] As can be seen from the figure, except for sugammadex sodium, the above-mentioned substances that may coexist have almost no effect on the fluorescence of CDs, indicating that the nano-CDs used in the present invention have excellent selectivity for the detection of sugammadex sodium.

[0055] Example 8 Accurately weigh 0.2178 g of sugammadex sodium bulk drug powder and dissolve it in 10 mL of water to obtain a 10 mmol / L sugammadex sodium mother solution, which is then diluted with water to obtain a 1 mmol / L sugammadex sodium solution.

[0056] Prepare 2 mmol / L ethylenediaminetetraacetic acid (EDTA) aqueous solution, 1 mmol / L γ-cyclodextrin aqueous solution, 8 mmol / L dimethyl sulfide aqueous solution and 3.2 mmol / L glucose aqueous solution.

[0057] The above solutions were mixed with the nano-CDs aqueous solution prepared in Example 1 at a volume ratio of 1:9 at room temperature for 5 minutes to obtain a mixed solution.

[0058] The sodium sugammadex solution in the mixed solution was replaced with an equal amount of water to obtain a diluted nano-CDs solution.

[0059] The fluorescence emission spectra of each mixed solution and the diluted solution of nano-CDs were tested. The results are shown in Figure 8 As can be seen from the figure, by comparing the fluorescence responses of the functional groups and structures in sodium sugammadex to nano-CDs, equimolar amounts of carboxyl (provided by EDTA), γ-cyclodextrin, thioether (provided by dimethyl sulfide), and hydroxyl (provided by glucose) were compared with sodium sugammadex. It can be seen that in addition to sodium sugammadex, EDTA also showed a certain degree of fluorescence enhancement, indicating that the fluorescence enhancement of sodium sugammadex on CDs originated from the effect of carboxyl groups and nano-CDs.

[0060] Example 9 The mother solution of the nano-CDs solution prepared in Example 1 was diluted with phosphate buffer solutions (the total concentration of phosphate was 0.1 mol / L) with different pH values ​​(pH 4, 5, 6, 7, 8, 9 and 10, respectively) to obtain nano-CDs phosphate buffer solutions with different pH values, all of which had a concentration of 0.28 mg / mL.

[0061] Accurately weigh 0.2178g of sodium sugammadex drug powder, dissolve it in 10mL of water to obtain 10mmol / L of sodium sugammadex mother liquor, and dilute it with water to obtain 1mmol / L of sodium sugammadex solution. The above sodium sugammadex solution was mixed with the above-mentioned nano-CDs phosphate buffer solutions of different pH values ​​at a volume ratio of 1:9 for 5 minutes at room temperature to obtain a mixed solution. Replace the sodium sugammadex solution in each mixed solution with an equal amount of water to obtain each nano-CDs diluted solution. Test the fluorescence intensity (F) of each mixed solution and the initial fluorescence intensity (F0) of the nano-CDs diluted solution, and calculate the fluorescence enhancement efficiency (F / F0) under the working environment of phosphate buffer solutions with different pH values, such as Fig. 9As shown in the figure, in phosphate buffer solutions with a pH of 4 to 10, sodium sugammadex (1 mmol / L) showed fluorescence enhancement on nano-CDs, and the fluorescence enhancement efficiency remained basically unchanged, indicating that the fluorescence detection method provided by the present invention is applicable to a wide pH range.

[0062] Example 10 0.2178 g of sodium sugammadex powder was accurately weighed and dissolved in 10 mL of water to obtain 10 mmol / L sodium sugammadex mother liquor, which was diluted with water to obtain 1 mmol / L sodium sugammadex solution. The sodium sugammadex solution was mixed with the nano-CDs aqueous solution prepared in Example 1 at a volume ratio of 1:9 at room temperature, and the fluorescence emission spectrum of the obtained mixed solution was tested at mixing times of 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min, respectively. The results are shown in FIG. Fig.10 As can be seen from the figure, after adding sodium sugammadex (1 mmol / L) and mixing for 5 to 30 minutes, the fluorescence enhancement efficiency of nano-CDs remained basically unchanged, indicating that this analytical detection method has no specific requirements for the reaction time and the test can be performed within 5 to 30 minutes after adding sodium sugammadex.

[0063] Embodiment 11 Clinical sugammadex sodium injection (labeled concentration of 200 mg / 2 mL) was diluted 100 times with water, and then mixed with the nano-CDs phosphate buffer solution prepared in Example 1 at a volume ratio of 1:9 at room temperature for 5 minutes to obtain a mixed solution. The sugammadex sodium solution in the mixed solution was replaced with an equal amount of water to obtain a nano-CDs diluted solution. The fluorescence emission spectra of the nano-CDs diluted solution and the mixed solution were tested respectively. Fig.11 As shown in the figure, the horizontal axis is the emission wavelength and the vertical axis is the fluorescence intensity. The emission spectrum of nano-CDs (CDs, solid line in the figure) shows that the fluorescence intensity is significantly enhanced after adding the sugammadex sodium injection sample (CDs+injection sample, dotted line in the figure), indicating that the sample contains sugammadex sodium.

[0064] According to the fluorescence intensity (F) of the mixed solution to be tested and the initial fluorescence intensity (F0) of the nano-CDs diluted solution, the fluorescence enhancement efficiency = F / F0 is calculated, and the linear regression equation of fluorescence enhancement efficiency-sugammadex sodium concentration obtained in Example 6 is substituted to obtain the concentration of sugammadex sodium in the sample solution to be tested, and the calculated result is 1.05 g / L. It can be calculated by the dilution multiple that the content of sugammadex sodium in the injection is 105 g / L, and the recovery rate is 105%. The test concentration of the present invention is close to its labeled concentration, indicating that the fluorescence detection method provided by the present invention has high accuracy.

[0065] Example 12 The only difference from Example 2 is that the concentration of the nano-CDs aqueous solution is adjusted from 0.28 mg / mL to 0.056 mg / mL. Fig.12 As shown, nano-CDs exhibited fluorescence emission under 365nm excitation, with an emission wavelength of 460nm (CDs, solid line in the figure). After adding sugammadex sodium solution (0.1mmol / L), the fluorescence intensity was significantly enhanced (CDs+SUG, dotted line in the figure), indicating that sugammadex sodium has the property of enhancing the fluorescence of nano-CDs.

[0066] Embodiment 13 The only difference from Example 2 is that the concentration of the nano-CDs aqueous solution is adjusted from 0.28 mg / mL to 0.56 mg / mL. Fig.13 As shown, nano-CDs exhibited fluorescence emission under 365nm excitation, with an emission wavelength of 460nm (CDs, solid line in the figure). After adding sugammadex sodium solution (0.1mmol / L), the fluorescence intensity was significantly enhanced (CDs+SUG, dotted line in the figure), indicating that sugammadex sodium has the property of enhancing the fluorescence of nano-CDs.

[0067] Comparative Example 1 Preparation of CDs fluorescent probe without nitrogen atoms: Weigh 2.000 g of citric acid powder in a beaker, spread it evenly and place it in a heating mantle at 200 o After pyrolysis reaction at 400 °C for 30 min, 2 mL of 10 mg / mL sodium hydroxide solution was added, and after ultrasonic dissolution, the mixture was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with water for 24 h to obtain a nitrogen-free CDs fluorescent probe mother solution, which was diluted with water to obtain a nitrogen-free CDs fluorescent probe aqueous solution with a concentration of 0.28 mg / mL.

[0068] The difference between the fluorescence detection and Example 2 is that the 0.28 mg / mL nano-CDs aqueous solution prepared in Example 1 is replaced with the 0.28 mg / mL CDs fluorescent probe aqueous solution without nitrogen atoms prepared in this comparative example. Fig.14 As shown, the CDs fluorescent probe without nitrogen atoms exhibits fluorescence emission under 365nm excitation, and the emission wavelength is 490nm (CDs, solid line in the figure). After adding sugammadex sodium solution (0.1mmol / L), the fluorescence intensity is not enhanced (CDs+SUG, dotted line in the figure), indicating that sugammadex sodium has no fluorescence enhancement response to the CDs fluorescent probe prepared in this comparative example.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of nano-CDs as fluorescent probes in the fluorescence detection of sugammadex sodium, wherein the nano-CDs contain nitrogen atoms.

2. The use according to claim 1, characterized in that: The particle size of the nano-CDs is less than 5 nm.

3. The use according to claim 1 or 2, characterized in that: The raw materials for preparing the nano CDs include polyethyleneimine and bromophenol blue.

4. The use according to claim 3, characterized in that: The preparation method of the nano-CDs comprises the following steps: mixing polyethyleneimine, bromophenol blue and water, and performing a hydrothermal reaction to obtain the nano-CDs.

5. A fluorescence detection method for sugammadex sodium, characterized in that: The following steps are involved: Perform fluorescence detection on the sample probe mixture and the probe solution to be tested respectively to obtain the fluorescence intensity to be tested and the initial fluorescence intensity; The sample-probe mixture to be tested includes a sugammadex sodium sample to be tested, nano-CDs and an aqueous phase, and the probe solution is different from the sample-probe mixture to be tested in that it does not contain the sugammadex sodium sample to be tested; Obtaining the fluorescence enhancement efficiency of the sugammadex sodium sample to be tested according to the ratio of the fluorescence intensity to be tested to the initial fluorescence intensity; The quantitative detection result of sugammadex sodium is obtained according to the fluorescence enhancement efficiency of the sugammadex sodium sample to be tested and the standard curve; the standard curve is a standard curve with the concentration of sugammadex sodium as the independent variable and the fluorescence enhancement efficiency as the dependent variable.

6. The fluorescence detection method according to claim 5, characterized in that: The test conditions of the fluorescence detection include: the fluorescence excitation wavelength is 365nm; the fluorescence emission wavelength detection range is 380-540nm; the slit width is 5nm; the fluorescence intensity at 460nm is the fluorescence intensity to be measured or the initial fluorescence intensity.

7. The fluorescence detection method according to claim 5, characterized in that: The linear range of the standard curve is 0.001-0.07 or 0.07-0.5 g / L.

8. The fluorescence detection method according to claim 5, characterized in that: The aqueous phase includes water or a phosphate buffer solution.

9. The fluorescence detection method according to claim 8, characterized in that: The pH value of the phosphate buffer solution is 4-10.

10. The fluorescence detection method according to claim 5, 8 or 9, characterized in that: The phosphate in the phosphate buffer solution includes at least one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, and diammonium hydrogen phosphate; The total concentration of phosphate in the phosphate buffer solution is 0.01-0.1 mol / L.

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