CN116879253B - A method for detecting Au(III) and creatinine using an "off-on" type fluorescence sensor - Google Patents
A method for detecting Au(III) and creatinine using an "off-on" type fluorescence sensorInfo
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Abstract
本发明公开了一种以“关‑开”型荧光传感器检测Au(Ⅲ)和肌酐的方法，属于Au(Ⅲ)和肌酐的分析检测领域。本发明检测方法的步骤如下：(1)以邻苯二胺和异丙醇为前驱体制备碳点；(2)碳点中Au3+的加入会通过静态猝灭和光致电子转移使荧光猝灭，基于此构建Au3+浓度和荧光猝灭程度的标准曲线并得到湖水样品中Au3+的浓度；(3)肌酐与尿液混合后加入到碳点和Au3+的体系中，构建肌酐浓度与荧光恢复程度的标准曲线并得到尿液样品中肌酐的浓度。本发明首次以“关‑开”型荧光传感器先后对Au3+和肌酐两种物质定量检测，且操作简单、安全高效。
This invention discloses a method for detecting Au(III) and creatinine using an "off-on" type fluorescence sensor, belonging to the field of analysis and detection of Au(III) and creatinine. The steps of the detection method of this invention are as follows: (1) Carbon dots are prepared using o-phenylenediamine and isopropanol as precursors; (2) The addition of Au 3+ to the carbon dots will quench the fluorescence through static quenching and photoinduced electron transfer. Based on this, a standard curve of Au 3+ concentration and fluorescence quenching degree is constructed to obtain the concentration of Au 3+ in the lake water sample; (3) Creatinine is mixed with urine and added to the system of carbon dots and Au 3+ . A standard curve of creatinine concentration and fluorescence recovery degree is constructed to obtain the concentration of creatinine in the urine sample. This invention is the first to use an "off-on" type fluorescence sensor to quantitatively detect two substances, Au 3+ and creatinine, and the operation is simple, safe and efficient.
Description
Technical Field
The invention relates to a method for detecting Au (III) and creatinine by using an off-on fluorescent sensor, belonging to the field of analysis and detection of Au (III) and creatinine.
Background
Kidneys play a vital role in filtering large amounts of metabolic waste products from the human body. In fact, creatinine (Cre) is a metabolic waste molecule in our muscle metabolic process. It is actually produced by creatine, which is used to produce the main energy in our muscles. Almost 2% of the precursor creatine is converted to creatinine daily and then transported through the blood to the kidneys. Creatinine levels in urine may rise when kidney function is abnormal. In early stages, the frequent detection of creatinine in human urine can improve the quality of life of people, especially for diabetics. There is therefore a great need in the field of early clinical diagnosis of these diseases to develop simple and low cost methods for the selective and sensitive detection of creatinine in biological samples. In addition, with the development of modern industry, heavy metal ion pollution seriously threatens natural environment and human health safety, and due to the strong coordination between Au 3+ ions and enzymes, the liver, kidney and nervous system of human body can be damaged. Therefore, it is essential to develop a method that is efficient, sensitive, has good specificity and can detect gold ions.
In recent years, various analytical methods for detecting creatinine have been developed, mainly high performance liquid chromatography, electrochemical methods, chemiluminescent methods and the like, which have problems of poor selectivity, high toxicity, low sensitivity, complex operation and the like, although they have high accuracy and reliability. Therefore, there is an urgent need to find a simpler, faster, and more sensitive Cre detection method.
Disclosure of Invention
Technical problems:
Various analytical methods for detecting creatinine developed at present mainly comprise high performance liquid chromatography, electrochemical methods, chemiluminescence methods and the like, and the methods have the problems of poor selectivity, high toxicity, low sensitivity, complex operation and the like. Meanwhile, the report of detecting creatinine in urine by using a fluorescence sensing technology is less, and the sensitivity and the detection limit are not advantageous compared with the traditional method.
The technical scheme is as follows:
A method for detecting Au 3+ and creatinine based on an off-on fluorescence sensor, comprising the steps of:
(1) Synthesizing carbon dots by a hydrothermal method by taking o-phenylenediamine and isopropanol as precursors, purifying and diluting the carbon dots to obtain a carbon dot solution for subsequent detection;
(2) Preparing Au 3+ standard solutions with different concentrations, uniformly mixing the Au 3+ standard solutions with carbon dot solution, phosphate buffer solution and lake water sample, fixing the volume by deionized water, incubating to obtain mixed test sample solution, and performing fluorescence spectrum detection;
(3) Respectively measuring the fluorescence intensity of the mixed test sample liquid with the concentration of 0 and the concentration of other than 0 of the Au 3+ standard solution, obtaining the corresponding fluorescence quenching degree by calculating the ratio of the fluorescence intensity of the mixed test sample liquid with the concentration of 0 and the concentration of other than 0 of the Au 3+ standard solution, and constructing a linear Au 3+ quantitative model by utilizing the fluorescence quenching degree and the concentration of Au 3+ in the corresponding mixed test sample liquid;
(4) Obtaining a lake water sample to be detected according to the step (2), detecting the lake water sample to be detected by a fluorescence spectrum to obtain the fluorescence intensity of the lake water sample to be detected, calculating to obtain the corresponding fluorescence quenching degree, and obtaining the Au 3+ concentration in the lake water sample to be detected according to the linear Au 3+ quantitative model in the step (3);
(5) Preparing creatinine standard solutions with different concentrations, mixing the creatinine standard solutions with a urine sample, adding a mixed solution of a carbon dot solution, a phosphate buffer solution and an Au 3+ standard solution, uniformly mixing, fixing the volume by deionized water, incubating to obtain a mixed urine test sample solution, and performing fluorescence spectrum detection;
(6) Respectively measuring the fluorescence intensity of the mixed urine test sample liquid with the concentration of the creatinine standard solution being different from 0 and the concentration of the mixed urine test sample liquid being different from 0, obtaining the corresponding fluorescence recovery degree by calculating the ratio of the fluorescence intensity of the mixed urine test sample liquid with the concentration of the creatinine standard solution being different from 0 and the concentration of the creatinine standard solution being 0, and constructing a linear creatinine quantitative model by utilizing the fluorescence recovery degree and the creatinine concentration in the corresponding mixed urine test sample liquid;
(7) And (3) obtaining a urine sample to be detected according to the step (5), detecting the urine sample to be detected by a fluorescence spectrum to obtain the fluorescence intensity of the urine sample to be detected, calculating to obtain the corresponding fluorescence recovery degree, and obtaining the concentration of creatinine in the urine sample to be detected according to the linear creatinine quantitative model in the step (6).
In one embodiment of the invention, in the step (1), the amount of the o-phenylenediamine to the isopropanol is 20-50mg/mL, and particularly, 40mg/mL is selected.
In one embodiment of the invention, in the step (1), o-phenylenediamine and isopropanol are added into deionized water for hydrothermal reaction, wherein the volume ratio of the isopropanol to the deionized water is 1 (3-8), and the specific selection is 1:4.
In one embodiment of the invention, in step (1), the hydrothermal reaction is carried out at a temperature of 150-200 ℃ for a time of 8-15 hours. The reaction is carried out for 10h at 180 ℃.
In one embodiment of the present invention, in the step (1), the synthesis process of the carbon dots specifically includes:
the method comprises the steps of adding o-phenylenediamine and isopropanol into deionized water, carrying out ultrasonic treatment for 10min to enable the mixture to be fully dissolved, and then reacting for 10h at 180 ℃, wherein the dosage of the o-phenylenediamine relative to the isopropanol is 40mg/mL, and the volume ratio of the isopropanol to the deionized water is 1:4.
In one embodiment of the invention, in the step (1), the purification process comprises the steps of cooling after the hydrothermal reaction is finished to obtain a crude carbon dot solution, centrifuging the crude carbon dot solution at 10000rpm for 10min, filtering to remove unreacted particles through a microporous filter membrane with the molecular weight cutoff of 1000 mu M, and finally dialyzing and purifying the carbon dot for 12h by using a dialysis membrane with the molecular weight cutoff of 1000Da to obtain a final carbon dot.
In one embodiment of the present invention, in the step (1), the dilution factor of the purified carbon dots is 100 to 200 to obtain a carbon dot solution. Specifically, the dilution is optionally 150 times.
In one embodiment of the invention, the conditions for fluorescence spectrum detection in the steps (2) and (5) are that the fluorescence spectrum is measured by a fluorescence spectrometer, the width of an excitation slit and an emission slit of the spectrometer is 3.0nm, the integration time is 0.1s, the excitation wavelength of the fluorescence spectrometer is 420nm, the emission wavelength ranges from 450nm to 750nm, and the step length is 1nm.
In one embodiment of the invention, in the step (2), the volume ratio of the carbon dot solution, the phosphate buffer solution, the lake water sample and the Au 3+ standard solution with different concentrations is 1:1:1:1.
In one embodiment of the invention, in the step (2), the volume-fixing condition is that the ratio of the volume after volume fixing to the volume of the Au 3+ standard solution is 10:1.
In one embodiment of the invention, in step (2), the incubation time is 1-20min, specifically 5min.
In one embodiment of the present invention, the linear Au 3+ quantitative model described in step (3) is:
F 0/F＝1.0582c(Au3+) -4.5696, wherein F 0 and F represent the fluorescence intensities of the mixed test sample solutions with 0 and non-0 concentrations of the Au 3+ standard solution, respectively, and c (Au 3+) represents the concentration of Au 3+ in the mixed test sample solution.
In one embodiment of the present invention, the volume ratio of the sample to be tested and the carbon dot solution in step (4) is 1:1.
In one embodiment of the present invention, the concentration of the Au 3+ standard solution in step (5) is fixed at 10. Mu. Mol/L.
In one embodiment of the invention, in the step (5), the volume ratio of the carbon dot solution, the phosphate buffer solution, the lake water sample and the Au 3+ standard solution with different concentrations is 1:1:1:1.
In one embodiment of the invention, in the step (5), the volume-fixing condition is that the ratio of the volume after volume fixing to the volume of the Au 3+ standard solution is 10:1.
In one embodiment of the invention, in step (5), the incubation time is 1-20min.
In one embodiment of the invention, the linear creatinine quantitative model in the step (6) is F/F 1 = 1.8536c (Cre) +1.1356, wherein F 1 and F respectively represent the fluorescence intensity of the mixed urine test sample liquid with the concentration of the creatinine standard solution being 0 and the concentration of the creatinine in the mixed urine test sample liquid being different from 0.
The invention also provides application of the detection method in the field of biomedical detection.
The beneficial effects are that:
The invention applies the off-on fluorescent sensor to the detection of Au 3+ and creatinine, the appearance of the carbon dots is spherical or spheroid, the surface of the carbon dots contains rich functional groups, and the water solubility of the carbon dots and the binding capacity with Au 3+ are improved.
According to the method, the carbon points synthesized by taking o-phenylenediamine and isopropanol as raw materials are used as the fluorescence sensor for the first time to sequentially realize quantitative detection of Au 3+ in lake water and creatinine in urine, and the method is simple, quick and safe to operate and suitable for routine analysis. In the method, au 3+ interacts with the functional groups on the surface of the carbon dots and generates electron transfer to cause fluorescence quenching, and meanwhile, the method also has the function of static quenching, has higher selectivity on Au 3+, has strong binding capacity on creatinine molecules and reduced gold nanoparticles, and then recovers fluorescence, and the sensor can sequentially detect Au 3+ and creatinine in the same system.
The linear interval of the detection Au 3+ is 8-12 mu mol/L, the detection limit is as low as 1.69 multiplied by 10 -8 mol/L, the linear interval of the detection creatinine is 0.05-1 mu mol/L, and the detection limit is 9.29 multiplied by 10 -9 mol/L, which has important significance for the fields of environmental monitoring and biomedicine.
Drawings
FIG. 1 is a flow chart for detecting Au 3+ and creatinine using an "off-on" fluorescence sensor.
FIG. 2 is a graph showing fluorescence spectra of Au 3+ added to the system of example 1 at different concentrations.
FIG. 3 is a graph showing the relationship between the fluorescence quenching degree and the Au 3+ concentration in example 1.
FIG. 4 is a linear fit of the degree of fluorescence quenching versus Au 3+ in the concentration range of 8-12. Mu.M in example 1.
FIG. 5 is a graph showing fluorescence spectra of the system of example 2 when creatinine was added at various concentrations.
FIG. 6 is a graph showing the relationship between the degree of fluorescence recovery and the concentration of creatinine in example 2.
FIG. 7 is a linear fit of the extent of fluorescence recovery versus creatinine in the concentration range of 0.05-1. Mu.M in example 2.
FIG. 8 is a graph showing the results of the test for detecting the selectivity of Au 3+ in example 3.
FIG. 9 is a graph showing the results of the test for creatinine selectivity in example 4.
FIG. 10 is a graph showing the results of the test for the effect of Au 3+ concentration on creatinine detection in example 5.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.
EXAMPLE 1 preparation of carbon Point (CDs) solutions
0.2G of o-phenylenediamine and 5mL of isopropanol were weighed into a beaker, 20mL of deionized water was added, the mixture was sonicated for 10min to dissolve thoroughly, and then reacted at 180℃for 10h, and cooled to obtain a crude carbon dot solution. Centrifuging the crude carbon spot solution at 10000rpm for 10min, collecting supernatant, filtering with 0.22 μm microporous membrane to remove unreacted particles, and dialyzing and purifying the carbon spot with dialysis membrane with molecular weight cut-off of 1000Da for 12 hr to obtain final carbon spot. The carbon dot solutions of the subsequent experiments are all based thereon.
EXAMPLE 2 construction of a Linear measurement model of Au 3+
(1) Sample solutions were prepared of carbon dot solution (150-fold dilution from the carbon dot obtained in example 1), phosphate buffer (ph=8), lake water sample and Au 3+ standard solutions with concentrations of 0 (blank), 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 9.5 μm, 10 μm, 11 μm, 11.5 μm, 12 μm, 13 μm, 14 μm, 15 μm, respectively;
(2) Mixing the four substances, wherein the volumes of a carbon dot solution, a phosphate buffer solution, a lake water sample and Au 3+ standard solutions with different concentrations are all 0.3mL, then using deionized water to fix the volume to 3mL, standing and incubating for 5min to obtain mixed test sample solutions with different Au 3+ concentrations, and performing fluorescence spectrum detection, wherein the reaction temperature is 20 ℃;
(3) Measuring fluorescence spectrum of the system under the scanning conditions that excitation wavelength is 420nm, emission wavelength scanning range is 450-750nm, scanning is carried out every 1nm, slit width is set to be 3.0nm/3.0nm (excitation slit/emission slit), fluorescence intensity peak value F (figure 2) at 420nm is obtained, mixed test sample liquid with Au 3+ concentration of 0 is added for fluorescence spectrum detection, fluorescence intensity peak value F 0 at 570nm is obtained, and fluorescence quenching degree F 0/F is recorded;
(4) The relation curve of the fluorescence quenching degree of the sample solution and the concentration of Au 3+ in the mixed test sample solution is drawn, as shown in fig. 3, the fitting curve of the quenching degree and Au 3+ is shown in fig. 4, and when the concentration of Au 3+ is 8-12 mu M, the fluorescence quenching degree of the solution and the concentration of Au 3+ are in a linear relation, the linear equation is F 0/F＝1.0582c(Au3+) -4.5696, the correlation coefficient is R 2 = 0.9911, and the detection limit is 1.69 multiplied by 10 -8 mol/L.
EXAMPLE 3 construction of a Linear measurement model of creatinine
(1) Sample solutions were prepared of carbon spot solution (150-fold dilution), phosphate buffer (ph=8), au 3+ standard solution (10 μm), urine sample and creatinine standard solutions with concentrations of 0 (blank), 0.05 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.7 μm, 0.8 μm, 1 μm, 1.5 μm, 2 μm, respectively;
(2) Firstly, uniformly mixing a carbon dot solution, a phosphate buffer solution and an Au 3+ standard solution, wherein the volumes of the carbon dot solution, the phosphate buffer solution and the Au 3+ standard solution are 0.3mL, then adding creatinine urine solutions with different concentrations (the volumes of creatinine and urine are 0.3 mL), finally, using deionized water to fix the volume to 3mL, standing and incubating for 20min to obtain mixed urine test sample solutions with different creatinine concentrations and adding labels, and carrying out fluorescence spectrum detection;
(3) Determining fluorescence spectrum of the system, wherein scanning conditions are consistent with detection Au 3+, adding a sample solution with creatinine concentration of 0 for fluorescence spectrum detection to obtain a fluorescence intensity peak value F 1 at 570nm, adding mixed urine test sample solution of other creatinine standard solutions with concentration of not 0 to obtain a fluorescence intensity peak value F, and recording fluorescence recovery degree F/F 1, as shown in figure 5;
(5) The relationship curve of the fluorescence recovery degree of the sample solution and the creatinine concentration in the mixed urine sample solution is drawn, as shown in fig. 6, the fitting curve of the recovery degree and the creatinine is shown in fig. 7, and when the creatinine concentration is 0.05-1 mu M, the fluorescence recovery degree of the solution and the creatinine concentration are in a linear relationship, the linear equation is F/F 1 = 1.8536c (Cre) +1.1356, the correlation coefficient is R 2 =0.9965, and the detection limit is 9.29×10 -9 mol/L.
Example 4 exploration of fluorescence "off" procedure to detect the selectivity of Au 3+
Referring to example 2, 12 different common metal ions (Na+、K+、Ca2+、Mg2+、Al3+、Cu2+、Fe3+、Zn2+、Hg2+、Pb2+、Ag+) were selected as interfering substances, and as shown in fig. 8, F 0 and F represent fluorescence intensities before and after the metal ions were added, respectively. All cation concentrations were 12. Mu.M and all fluorescence measurements were performed under the same conditions.
According to the detection result, the interference substance has no obvious fluorescence quenching effect on carbon points, and does not interfere detection of Au 3+ ions.
Example 5 exploration of the fluorescence "on" procedure to detect creatinine selectivity
Referring to example 3, the selectivity of the CDs-Au 3+ system for detecting creatinine was studied using eight common organic and inorganic salt ions (urea, uric acid, glucose, ascorbic acid, dopamine, histidine, cl -、NO3 -) in urine as interferents, the concentration of creatinine and interferents were 10 μm, and the concentration of Au 3+ was fixed at 10 μm. The detection results are shown in FIG. 9.
From the images, creatinine has obvious recovery effect on fluorescence of the CDs-Au 3+ system, and other substances can not recover the fluorescence basically.
Example 6 investigation of the Effect of Au 3+ concentration in creatinine detection
Referring to example 3, au 3+ standard solutions of different concentrations (8 μm, 9 μm, 10 μm, 11 μm, 12 μm) were added to the carbon dot solution, and in order to maximize the sensitivity of detecting creatinine, the concentration of creatinine was controlled to 0.5 μm, and the measured result was as shown in fig. 10, and when Au 3+ concentration was 10 μm, the fluorescence recovery effect was slightly remarkable, and thus, au 3+ concentration was fixed at 10 μm at the time of detecting creatinine.
Example 7 detection of Au in lake Water Environment 3+
Referring to example 2, au 3+ at a concentration of 8,9, 10. Mu.M was measured and the detection results are shown in Table 1.
Table 1 test results of example 7
| Concentration of addition mark (mu M) | Concentration detection (mu M) | Recovery (%) | Relative standard deviation (%), n=3 |
| 8 | 8.06 | 100.7 | 0.33 |
| 9 | 8.97 | 99.7 | 0.31 |
| 10 | 10.06 | 100.6 | 0.58 |
Example 8 detection of creatinine in urine environment
Referring to example 3, creatinine with a concentration of 100,300,500nM was measured, and the results are shown in Table 2, and the results are satisfactory, which indicates that the method is accurate and feasible and can be applied to the biomedical field for creatinine detection.
Table 2 test results of example 8
| Concentration of addition standard (nM) | Detection concentration (nM) | Recovery (%) | Relative standard deviation (%), n=3 |
| 100 | 101 | 101.0 | 0.79 |
| 300 | 292 | 97.3 | 0.75 |
| 500 | 527 | 105.4 | 2.11 |
Comparative example 1 comparison of creatinine detection by other fluorescence methods in the prior report
In the prior report (Label-free detection of creatinine using nitrogenpassivated fluorescent carbon dots.RSC Adv.2020,10,36253), the carbon point is synthesized by using ascorbic acid, urea, thiourea and cysteine as raw materials, and then picric acid is used for modification, wherein picric acid is an explosive substance, and the tube product is not suitable for being used as a synthetic material.
Meanwhile, (A copper nanoclusters probe for dual detection of microalbumin and Creatinine.Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy.2022,270,120816) is reported to detect creatinine by using copper nanoclusters, but the detection limit of the method is only 1.3X10 -5 M, and the method has no advantage for detecting trace creatinine. Therefore, the method has the advantages of rapid preparation, sensitive detection and low cost.
Comparative example 2 preparation of carbon dots from different carbon sources and o-phenylenediamine
0.2G of o-phenylenediamine and 5mL of glycerin were weighed into a beaker, 20mL of deionized water was added, and the mixture was sonicated for 10min to dissolve thoroughly, followed by reaction at 180℃for 10h. The synthesized crude carbon dot solution was centrifuged at 10000rpm for 10min, and then unreacted particles were removed by filtration through a microporous membrane of 0.22. Mu.M, and finally the carbon dot was dialyzed and purified with a dialysis membrane having a molecular weight cut-off of 1000Da for 12 hours to obtain the final carbon dot.
The carbon dots obtained in example 1 and comparative example 2 were diluted 150 times and subjected to fluorescence spectrum detection under excitation light with a wavelength of 420nm, the fluorescence peak positions of the two were different, 0.3mL of each of the two carbon dots was taken and placed in a test tube, 0.3mL of 10. Mu.M Au 3+ solution and 0.3mL of 1. Mu.M Cre solution were added, the volume was fixed to 3mL with deionized water, and fluorescence spectrum detection was performed after incubation for 3 min. The fluorescence quenching rate and the fluorescence recovery rate in the carbon dot system obtained in example 1 were higher than those in comparative example 2.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
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