A Method for Dual-Ratio Fluorescence Detection of Morphine in Blood Using Nitrogen-Doped Carbon Quantum Dots

Abstract

The present invention discloses a method for dual-ratio fluorescence detection of morphine in blood using nitrogen-doped carbon quantum dots; the present invention uses m-phenylenediamine and spermidine as carbon and nitrogen sources, and synthesizes nitrogen-doped fluorescent carbon quantum dots with high fluorescence quantum yield by microwave method; due to the fluorescence resonance energy transfer effect between morphine and carbon quantum dots, in the presence of morphine, the fluorescence of carbon quantum dots decreases at 350 nm, while increases sharply at 456 nm, and the ratio of the fluorescence intensity at 350 nm to that at 456 nm has a certain linear relationship with the morphine concentration, based on which a fluorescence spectrophotometry for detecting morphine in blood is established; the nitrogen-doped fluorescent carbon quantum dots prepared by the present invention have significant fluorescence sensitization and quenching effects on morphine, and have strong selectivity, and are used for the fluorescence determination of morphine in blood, with the characteristics of high sensitivity and specificity.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical analysis and detection, and particularly relates to a method for detecting morphine by double-ratio fluorescence of nitrogen-doped carbon quantum dots. Background Art

[0002] The drug problem is closely related to people's production and life. Solving the drug problem not only helps people's living health, but also contributes to social stability. In the processes of identifying drug addicts and monitoring the drug rehabilitation treatment process, various drug detection technologies play an important role. Morphine is an important product of the hydrolysis of heroin and other drugs in vivo, and is an important indicator for detecting the drug content in forensic cases. To prevent the drug problem and the toxicity of drug overdose, rapid and quantitative detection of the morphine concentration in blood is a key technology in clinical and forensic identification. The commonly used methods for determining its content include high performance liquid chromatography, gas chromatography, high performance liquid chromatography-mass spectrometry, and gas chromatography-mass spectrometry. These detection methods not only require professional staff to process the samples, but also require large and expensive detection instruments, which limits the time for on-site drug identification.

[0003] Carbon quantum dots are a new type of nanomaterial composed of carbon atoms with a particle size less than 10 nm. They have good optical properties, adjustable spectral properties, long fluorescence lifetimes, and good biocompatibility. They have been widely used in the fields of biology, medicine, catalysis, optoelectronics, bioimaging, and analysis and detection, and have gradually become a rising star in nanomaterials. Currently, researchers are committed to exploring simple, green, and low-cost methods for preparing carbon dots with high fluorescence quantum yields, and significantly improving their fluorescence quantum yields and optoelectronic properties through doping or surface passivation. In recent years, there have been many literature reports on the research of nitrogen-doped carbon quantum dots, and the fluorescence quantum yields of carbon quantum dots have been further improved; the highly sensitive and highly selective technical solution provided by the present invention has not been reported yet. Summary of the Invention

[0004] The present invention provides a method for detecting morphine in blood by double-ratio fluorescence of nitrogen-doped carbon quantum dots with high sensitivity and high selectivity; the nitrogen-doped fluorescent carbon quantum dots prepared by the present invention have a significant fluorescence sensitization and quenching effect on morphine, and have strong selectivity. They are used for the fluorescence determination of morphine in blood and have the characteristics of high sensitivity and specificity.

[0005] The method for detecting morphine in blood by double-ratio fluorescence of nitrogen-doped carbon quantum dots of the present invention is as follows:

[0006] (1) Preparation of water-soluble nitrogen-doped carbon quantum dots

[0007] 0.3-0.5 g of m-phenylenediamine and 0.3-0.5 g of spermidine were added to 40 mL of ultrapure water, shaken to dissolve, and then heated in a microwave at 160-200 ° C for 1-2 h. After natural cooling, the mixture was filtered with a filter membrane with a pore size of 0.22 μm, and then dialyzed with a dialysis bag with a molecular weight cutoff of 3000-3500 Da for 24 h. The dialyzate was collected to obtain a water-soluble nitrogen-doped carbon quantum dot solution;

[0008] (2) Drawing of standard working curve

[0009] A water-soluble nitrogen-doped fluorescent carbon quantum dot solution was added to a morphine standard solution, and the pH was adjusted to 8. The solution was then made up to volume with deionized water to prepare a morphine standard solution with a concentration in the range of 0.25-25 μg / mL. The solution was vortexed and allowed to stand. The fluorescence intensity of the mixture was measured using a fluorescence spectrophotometer at an excitation wavelength of 310 nm and an emission wavelength of 350 nm, and at an excitation wavelength of 310 nm and an emission wavelength of 456 nm, respectively. The ratio of the fluorescence intensity at 350 nm to the fluorescence intensity at 456 nm was calculated, and the linear relationship between the morphine concentration and the fluorescence intensity ratio was determined to obtain a standard working curve and a linear regression equation.

[0010] The standing is to place at 40-45° C. for 10-20 minutes; the amount of the water-soluble nitrogen-doped fluorescent carbon quantum dot solution is 80-120 μL;

[0011] (3) Sample preparation

[0012] Plasma was placed in a centrifuge tube, centrifuged at 4000 r / min for 30 min, and the supernatant was aspirated;

[0013] (4) Sample measurement

[0014] The supernatant of step (3) was added with a water-soluble nitrogen-doped fluorescent carbon quantum dot solution, the pH was adjusted to 8.0 with a citric acid-disodium hydrogen phosphate buffer solution and the volume was made up with water, the mixture was vortexed and allowed to stand, and the fluorescence intensity of the sample to be tested was detected using a fluorescence spectrophotometer at an excitation wavelength of 310 nm and an emission wavelength of 350 nm, and at an excitation wavelength of 310 nm and an emission wavelength of 456 nm, respectively. The ratio of the fluorescence intensity at 350 nm to the fluorescence intensity at 456 nm was calculated and substituted into the linear regression equation of step (2) to obtain the content of morphine in the sample to be tested;

[0015] The standing is to place at 40-45° C. for 10-20 minutes, and the amount of the water-soluble nitrogen-doped fluorescent carbon quantum dot solution used is 80-120 μL.

[0016] The advantages of the present invention are:

[0017] 1. The present invention uses m-phenylenediamine and spermidine to prepare nitrogen-doped carbon quantum dots with water solubility and good fluorescence properties by microwave method. The prepared nitrogen-doped carbon quantum dots have a high fluorescence quantum yield. The fluorescence quantum yield of the nitrogen-doped carbon quantum dots is as high as 12.95%. It has a dual effect of fluorescence quenching of morphine at 350 nm and fluorescence sensitization at 456 nm. Using the nitrogen-doped fluorescent carbon quantum dots as a fluorescence probe to detect trace morphine in blood, the method has high sensitivity, and other coexisting ions and analogs do not have this effect, and the method has good specificity.

[0018] 2. The nitrogen-doped fluorescent carbon quantum dots of the present invention can specifically quench and sensitize the dual-ratio fluorescence of morphine, and can be used for the rapid detection of morphine in blood. The method is simple, highly sensitive, and highly specific. Description of the Drawings

[0019] Figure 1 It is a linear relationship diagram of water-soluble nitrogen-doped fluorescent carbon quantum dots to morphine in Example 1;

[0020] Figure 2 It is a fluorescence scanning diagram of different concentrations of morphine solution interacting with water-soluble nitrogen-doped fluorescent carbon quantum dots;

[0021] Figure 3 It is the influence result of various coexisting ions on nitrogen-doped fluorescent carbon quantum dots;

[0022] Figure 4 It is the influence result of various coexisting amino acids on nitrogen-doped fluorescent carbon quantum dots. Detailed Embodiments

[0023] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto; reagents in the embodiments are conventional reagents unless otherwise specified, and methods are conventional methods unless otherwise specified;

[0024] Example 1: Determination of the content of morphine in blood

[0025] (1) Preparation of water-soluble nitrogen-doped fluorescent carbon quantum dots (CQDs)

[0026] Weigh 0.3 g of m-phenylenediamine and 0.5 g of spermidine, add them to 40 mL of ultrapure water, shake and dissolve, transfer them to a polytetrafluoroethylene reaction kettle, place them in a microwave digestion instrument, heat them by microwave to 160 °C for 2 h, cool naturally, filter with a filter membrane with a pore size of 0.22 μm, and then dialyze with a dialysis bag with a cut-off molecular weight of 3000 Da for 24 h. Collect the permeate to obtain water-soluble nitrogen-doped fluorescent carbon quantum dots;

[0027] (2) Drawing of the standard working curve

[0028] Eight concentrations of morphine standard solutions were prepared with deionized water. That is, 100 μL of water-soluble nitrogen-doped fluorescent carbon quantum dot solution was added to the morphine standard solution, and the pH was adjusted to 8.0 with citric acid-disodium hydrogen phosphate buffer solution, and then made up to 4 mL with water to obtain morphine standard solutions with concentrations of 0.25, 0.5, 1, 1.25, 2.5, 5, 10, and 25 μg / mL. Then, after vortexing and mixing respectively, they were left standing for 15 min in a 44 °C water bath. Using a fluorescence spectrophotometer, the fluorescence intensities of the mixtures were measured at an excitation wavelength of 310 nm and an emission wavelength of 350 nm, and at an excitation wavelength of 310 nm and an emission wavelength of 456 nm respectively. The ratio of the fluorescence intensity at 350 nm to the fluorescence intensity at 456 nm was calculated. With the morphine concentration as the abscissa and the fluorescence intensity ratio as the ordinate, a standard working curve was plotted, and a linear regression equation I 350 / I 456 = 1.6027 - 0.02622C was obtained, and the correlation coefficient R 2 = 0.991 (see Figure 1 );

[0029] From Figure 2 is the fluorescence scanning diagram of the interaction between morphine solutions with different concentrations and water-soluble nitrogen-doped fluorescent carbon quantum dots. It can be seen from the figure that as the morphine concentration increases, the fluorescence intensity of water-soluble nitrogen-doped fluorescent carbon quantum dots at 350 nm gradually weakens, while the fluorescence at 456 nm gradually increases;

[0030] (3) Plasma was placed in a centrifuge tube and centrifuged at 4000 r / min for 30 min, and the supernatant was aspirated;

[0031] (4) Take 20 μL of the supernatant from step (3), add 100 μL of water-soluble nitrogen-doped fluorescent carbon quantum dot solution, adjust the pH to 8.0 with citric acid-disodium hydrogen phosphate buffer solution and make up to 4 mL with water. After vortexing and mixing, it was left standing for 15 min in a 44 °C water bath. Using a fluorescence spectrophotometer, the fluorescence intensities of the blood samples were measured at an excitation wavelength of 310 nm and an emission wavelength of 350 nm, and at an excitation wavelength of 310 nm and an emission wavelength of 456 nm respectively. The ratio of the fluorescence intensity at 350 nm to the fluorescence intensity at 456 nm was calculated and substituted into the linear regression equation in step 2 to calculate the morphine content in the blood to be 3.7 μg / mL;

[0032] (5) Specific detection of water-soluble nitrogen-doped fluorescent carbon quantum dots

[0033] According to the method in step (2), the 5 μg / mL morphine standard solution was replaced with ascorbic acid, amino acids, and coexisting ions with a concentration of 100 μg / mL, and then water-soluble nitrogen-doped fluorescent carbon quantum dots (CQDs) were added to detect the specificity of the method system of the present invention. The results are shown in Figure 3 、Figure 4 As can be seen from the figure, various coexisting ions and amino acids do not interfere with the determination of morphine, and the method has good specificity;

[0034] (6)Determination of the quantum yield of nitrogen-doped fluorescent carbon quantum dots

[0035] Referring to the literature "Ratiometric fluorescence assay based on carbon dots and Cu 2+ -catalyzed oxidation of O -phenylenediamine for the effective detection ofdeferasirox” (RSC Advances, 2021, 11, 34525, DOI: 10.1039 / d1ra07078a), quinine sulfate was selected as the reference substance. The fluorescence quantum efficiency of quinine sulfate in 0.1 mol / L H2SO4 solution is 54.6%. Prepare a 0.1 mol / L quinine sulfate solution with 0.1 mol / L H2SO4, measure its ultraviolet absorption spectrum, and record its absorbance at 333 nm; use 310 nm as its excitation wavelength and 390 nm as its emission spectrum, and record its fluorescence intensity; prepare a 1.232 mg / L nitrogen-doped fluorescent carbon quantum dot solution with deionized water, measure its ultraviolet absorption spectrum, and record its absorbance at 343 nm; use 310 nm as its excitation wavelength and 350 nm as its emission spectrum, and record its fluorescence intensity; substitute the above values into the formula Q = Q R × (I S / I R ) × (A R / A S ) × (η S 2 / η R 2 ) , where: Q is the quantum yield, A R is the ultraviolet absorbance of quinine sulfate, A S is the ultraviolet absorbance of nitrogen-doped fluorescent carbon quantum dots, I R is the fluorescence intensity of quinine sulfate, I Sis the fluorescence intensity of nitrogen-doped fluorescent carbon quantum dots, Q R The fluorescence quantum efficiency of quinine sulfate is 54.6%, η is the refractive index of the solution (where η S = η R = 1.33), and the quantum yield of nitrogen-doped fluorescent carbon quantum dots is calculated to be 12.95%;

[0036] (7) Recovery and precision experiments

[0037] Morphine standard solutions with two concentrations were added to blood samples, and each concentration was determined in parallel 3 times. The spiked recovery rate was calculated, and the relative standard deviation RSD was calculated. The results are shown in Table 1. The spiked recovery rate of morphine was measured to be 93.85% - 103.28%, and the RSD was 3.55% - 5.62%. This method has good accuracy and precision;

[0038] Table 1 Detection data of recovery rate

[0039] .

[0040] Example 2: Determination of the content of morphine in blood. The specific operation steps are as follows:

[0041] (1) Preparation of water-soluble nitrogen-doped fluorescent carbon quantum dots (CQDs)

[0042] Weigh 0.4 g of m-phenylenediamine and 0.4 g of spermidine, add them to 40 mL of ultrapure water, shake to dissolve, transfer to a polytetrafluoroethylene reaction kettle, place it in a microwave digestion instrument, and heat it to 180 °C by microwave for 1.5 h. After natural cooling, filter with a filter membrane with a pore size of 0.22 μm, and then dialyze with a dialysis bag with a molecular weight cut-off of 3000 Da for 24 h. Collect the permeate to obtain water-soluble nitrogen-doped fluorescent carbon quantum dots;

[0043] (2) Drawing of the standard working curve: The same as step (2) of Example 1;

[0044] (3) Take plasma and place it in a centrifuge tube, centrifuge at 4000 r / min for 30 min, and aspirate the supernatant;

[0045] (4) Take 20 μL of the supernatant from step (3), add 80 μL of the water-soluble nitrogen-doped fluorescent carbon quantum dot solution, adjust the pH to 8.0 with citrate-disodium hydrogen phosphate buffer solution and make up the volume to 4 mL with water. Vortex and mix well, then let it stand for 20 min in a water bath at 44 °C. On a fluorescence spectrophotometer, measure the fluorescence intensity at an excitation wavelength of 310 nm and emission wavelengths of 350 nm and 456 nm. Substitute the ratio of the fluorescence intensity at 350 nm to the fluorescence intensity at 456 nm into the regression equation in step (2) to calculate the content of morphine in the blood. The content of morphine is obtained as 2.48 μg / mL, and the relative standard deviation is 5.3%. According to the recommendation of the International Union of Pure and Applied Chemistry (IUPAC), the detection limit is calculated as 71.8 ng / mL using 3s / k (s is the standard deviation of continuously measuring 10 blank solutions, and k is the slope of the linear regression equation).

[0046] Example 3: Determination of the content of morphine in blood. The specific operation steps are as follows:

[0047] (1) Preparation of water-soluble nitrogen-doped fluorescent carbon quantum dots (CQDs)

[0048] Weigh 0.5 g of m-phenylenediamine and 0.3 g of spermidine, add them to 40 mL of ultrapure water, shake and dissolve, then transfer to a polytetrafluoroethylene reaction kettle, place it in a microwave digestion instrument and heat it to 200 °C by microwave for 1 h. After natural cooling, filter with a filter membrane with a pore size of 0.22 μm, and then dialyze with a dialysis bag with a molecular weight cut-off of 3500 Da for 24 h. Collect the permeate to obtain water-soluble nitrogen-doped fluorescent carbon quantum dots.

[0049] (2) Drawing of the standard working curve: The same as step (2) in Example 1;

[0050] (3) Take the plasma and place it in a centrifuge tube, centrifuge at 4000 r / min for 30 min, and aspirate the supernatant;

[0051] (4) Sample determination: Take 20 μL of the supernatant from step (3), add 120 μL of the water-soluble nitrogen-doped fluorescent carbon quantum dot solution, adjust the pH to 8.0 with citrate-disodium hydrogen phosphate buffer solution and make up the volume to 4 mL with water. Vortex and mix well, then let it stand for 10 min in a water bath at 44 °C. On a fluorescence spectrophotometer, measure the fluorescence intensity at an excitation wavelength of 310 nm and emission wavelengths of 350 nm and 456 nm. Substitute the ratio of the fluorescence intensity at 350 nm to the fluorescence intensity at 456 nm into the regression equation in step (2) to calculate the content of morphine in the blood. The content of morphine is obtained as 20.67 μg / mL, and the relative standard deviation is 4.5%.

Claims

1. A method for dual-ratio fluorescence detection of morphine in blood using nitrogen-doped carbon quantum dots, characterized in that, The steps are as follows: (1) Using m-phenylenediamine and spermidine as carbon and nitrogen sources, synthesize a nitrogen-doped fluorescent carbon quantum dot solution under microwave conditions; (2) Add the nitrogen-doped fluorescent carbon quantum dot solution to a morphine standard solution with a concentration range of 0.25 - 25 μg / mL and a pH of 8, mix well and let stand. Then, use a fluorescence spectrophotometer to detect the fluorescence intensity of the mixture at an excitation wavelength of 310 nm and an emission wavelength of 350 nm, and at an excitation wavelength of 310 nm and an emission wavelength of 456 nm respectively. Calculate the ratio of the fluorescence intensity at 350 nm to the fluorescence intensity at 456 nm, and determine the linear relationship between the morphine concentration and the fluorescence intensity ratio to obtain the standard working curve and the linear regression equation; (3) Add the nitrogen-doped fluorescent carbon quantum dot solution to the sample solution to be tested with a pH of 8.0, mix well and let stand. Then, use a fluorescence spectrophotometer to detect the fluorescence intensity of the sample solution to be tested at an excitation wavelength of 310 nm and an emission wavelength of 350 nm, and at an excitation wavelength of 310 nm and an emission wavelength of 456 nm respectively. Calculate the ratio of the fluorescence intensity at 350 nm to the fluorescence intensity at 456 nm and substitute it into the linear regression equation in step (2) to obtain the content of morphine in the sample to be tested; The nitrogen-doped fluorescent carbon quantum dot solution is prepared by adding 0.3 - 0.5 g of m-phenylenediamine and 0.3 - 0.5 g of spermidine to 40 mL of ultrapure water, shaking to dissolve, heating at 160 - 200 °C under microwave for 1 - 2 h, naturally cooling, filtering with a filter membrane with a pore size of 0.22 μm, and then performing dialysis treatment for 24 h with a dialysis bag with a molecular weight cut-off of 3000 - 3500 Da.

2. The method for dual-ratio fluorescence detection of morphine in blood using nitrogen-doped carbon quantum dots according to claim 1, wherein: In steps (2) and (3), standing is carried out at 40 - 45 °C for 10 - 20 min.

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