CN110455753B - Non-enzymatic fluorescence sensing detection method for glucose in human urine - Google Patents
Non-enzymatic fluorescence sensing detection method for glucose in human urine Download PDFInfo
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Abstract
The invention discloses a non-enzymatic fluorescence sensing detection method for glucose in human urine. The fluorescent probe used by the sensing detection method is a nitrogen-doped carbon quantum dot. The nitrogen-doped carbon quantum dot solution is obtained by thermal polymerization of formamide, the temperature of solvothermal reaction is 120-220 ℃, the reaction time is 0.5-20 h, and ammonia gas is released by thermal polymerization; uniformly mixing the nitrogen-doped carbon quantum dot solution obtained in the process with urine containing glucose with different concentrations respectively, ensuring that the volumes of the nitrogen-doped carbon quantum dot solution and the urine are the same, testing the fluorescence emission intensities of the nitrogen-doped carbon quantum dot solution and the urine, and obtaining a detection limit and a linear range, wherein the excitation wavelength is 300-400 nm; and (3) mixing the urine to be detected and the nitrogen-doped carbon quantum dot solution in equal volume, and detecting the fluorescence intensity to obtain the concentration of the glucose. The non-enzymatic fluorescence sensing detection method provided by the invention can be used for quantitatively detecting glucose and has good anti-interference performance and selectivity.
Description
Technical Field
The invention relates to a non-enzymatic fluorescence sensing detection method for glucose in human urine, belonging to the technical field of analysis and detection.
Background
Develops a rapid, accurate and high-selectivity glucose detection method, and can be widely applied to the fields of clinical diagnosis, diabetes prevention, food monitoring, drug analysis, biochemical reaction process research and the like. Currently, glucose sensing detection methods are mainly classified as enzyme-based or non-enzyme-based sensors. Enzyme-based sensing assays are highly sensitive to the conditions of detection, because the enzymes used (e.g., glucose dehydrogenase and glucose oxidase) are sensitive to environmental pH, temperature and humidity, and thus are relatively unstable [ H. Zhu, L. Li, W. Zhou et al, Advances in non-enzymatic glucose sensors based on metal oxides, J. Mater. chem. B4 (46 (2016) 7333 ] 7349 ]. The stability of the non-enzyme-based sensing detection method is substantially improved compared with the enzyme-based sensing method; non-enzymatic sensing methods mainly focus on electrochemical sensing detection, and the detection principle of the methods is based on electrochemical oxidation of glucose by electrode materials, but high oxidation overpotential can cause corrosion of the electrode materials and electrode pollution caused by formed intermediate products, which all affect the accuracy of the detection.
Commercial glucose sensor technology is mature and mainly comprises two detection strategies: enzyme-based electrochemical sensors and light sensing methods based on light scattering. In addition to the above-mentioned drawbacks of enzyme-based sensors, the development of non-invasive, non-enzymatic sensing detection methods is of great importance since most commercial glucose sensors require the collection of blood samples and require cumbersome sample pre-treatment procedures. The fluorescence sensing method can realize non-invasive detection, and is not susceptible to electrolyte like the electrochemical sensing detection method, so the fluorescence sensing method is an ideal detection method for glucose [ M.S. Steiner, A. Duerkop, O.S. Wolfbeis, Optical methods for sensing glucose, chem. Soc. Rev. 40(9) (2011) 4805-4839 ].
The performance (e.g., selectivity, sensitivity, stability) of fluorescence sensing methods depends on the design of the fluorescent material or fluorescent probe. There are two modes of fluorescence sensing for detecting glucose: quenching of the modified fluorophore due to interaction of glucose with the probe molecule; based on quenching or enhancement of the fluorescence of the probe material itself. The former mode is commonly used in the field of biosensing, and the latter mode is mostly used in the field of chemical sensing. Compared with the biosensing detection method, the chemical sensing detection method based on the fluorescence enhancement or quenching of the probe material has better stability, and the influence of the detection environment (such as pH) on the sensing performance is small. The fluorescence chemical sensing detection method is mainly based on inorganic nano materials, particularly quantum dots such as carbon quantum dots, has good biocompatibility, chemical stability, fluorescence luminescence performance, enzyme-like property and other catalytic performance, and is widely applied to the field of fluorescence sensing. The carbon quantum dots are also easy to modify, such as doping elements can further improve the fluorescence emission performance. The reported fluorescence yield (phi) of nitrogen-doped carbon quantum dots or carbon-nitrogen-triple quantum dots reaches 0.3-0.9 [ J.Wu, S.W. Yang, J.P.Li, et al, Electron injection of phosphor doped g-C3N4 quantum dots: controllable photoluminescence emission wavelength in the whole visible light range with high quantum yield, Adv. Opt. Mater. 4(12) (2016) 2095-2101; M. C. Rong, Z. X. Cai, L. Xie, et al., Study on the ultrahigh quantum yield of fluorescent P, O-g-C3N4 nanodots and its application in cell imaging, Chem. Eur. J. 22(27) (2016) 9387-9395]. However, the above fluorescent materials are only used in buffers (such as NaOH and PBS) that are manually configured, and there is little consideration for practical scenes or applications in environments, such as blood or urine. Compared with glucose in blood, the method is more reliable in detecting glucose in urine, and meanwhile, invasive damage to a human body is avoided.
Disclosure of Invention
The purpose is as follows: in order to overcome the defects in the prior art, the invention provides a non-enzymatic fluorescence sensing detection method for detecting glucose in human urine; the nitrogen-doped carbon quantum dots with good stability are formed by thermal polymerization of formamide for the first time by using a solvothermal method, and fluorescence can be selectively quenched by glucose molecules, so that quantitative detection of glucose in urine is realized; the urine to be detected does not need to be pretreated, and the device has practical prospect.
The technical scheme is as follows: in order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a non-enzymatic fluorescence sensing detection method for glucose in human urine is based on the fluorescence emission performance of nitrogen-doped carbon quantum dots, and the fluorescence performance of the nitrogen-doped carbon quantum dots is selectively quenched by glucose, so that qualitative and quantitative detection of the glucose in the urine is realized.
Preferably, the non-enzymatic fluorescence sensing detection method for glucose in human urine comprises the following specific steps:
1) mixing urine of a healthy human body and a nitrogen-doped carbon quantum dot solution taking formamide as a solvent according to a volume ratio of 1:1, adding glucose solutions with different concentrations, and uniformly mixing to form a to-be-detected solution containing glucose with different concentrations;
2) exciting the liquid to be detected formed in the step 1) under the excitation of incident light with the wavelength of 300-400 nm, taking nitrogen-doped carbon quantum dots as fluorescent probes to generate fluorescence with different intensities, and obtaining the sensitivity and the linear range of the sensing detection method according to the relation between the concentration of glucose in urine and the fluorescence intensity;
3) for the detection of an actual sample, adding an equal volume of the nitrogen-doped carbon quantum dot solution into human urine to be detected, uniformly mixing, obtaining the fluorescence intensity of the sample to be detected by adopting the exciting light with the same wavelength in the step 2), and calculating the content of glucose in the urine according to the linear relation between the concentration of glucose and the fluorescence intensity in the step 2).
Preferably, the precursor of the nitrogen-doped carbon quantum dot is formamide, and a nitrogen-doped carbon quantum dot solution with formamide as a solvent is formed.
Preferably, the nitrogen-doped carbon quantum dot solution is prepared by the following method,
pouring a raw material formamide solvent into a polytetrafluoroethylene container, wherein the volume of the formamide solvent accounts for 60-80% of the volume of the container, the reaction temperature is 120-220 ℃, the reaction time is 0.5-20 h, ammonia gas is released by thermal polymerization to form a mixed solution of nitrogen-doped carbon quantum dots and formamide, and the mass concentration of the nitrogen-doped carbon quantum dots is 0.025-1% according to the difference of the time or temperature of the solvent thermal reaction.
Preferably, the lower the reaction temperature, the longer the reaction time when preparing the nitrogen-doped carbon quantum dots.
Preferably, the detection method has high selectivity on glucose and is derived from hydrogen bonds formed by dangling bonds at the edge of the nitrogen-doped carbon quantum dot and glucose molecules.
Preferably, the method for verifying the selective quenching of the fluorescence property of the nitrogen-doped carbon quantum dot by glucose is as follows,
the method comprises the steps of selecting four original solutions to be tested, namely urine, a mixed solution of urine and cysteine, a mixed solution of urine, albumin and creatinine, and a mixed solution of urine and glucose, adding nitrogen-doped carbon quantum dot solutions into the four original solutions to be tested respectively, and testing the fluorescence intensity of the four solutions to be tested under the excitation of light with the same wavelength.
Has the advantages that: the invention utilizes a hydrothermal method to synthesize the semiconductor nitrogen-doped carbon quantum dots with good water solubility in one step, and the preparation method is simple and reliable. The nitrogen-doped carbon quantum dot has good fluorescence emission performance, and the fluorescence of the nitrogen-doped carbon quantum dot can be selectively quenched by glucose molecules. Based on the characteristics, the method is applied to the quantitative detection of glucose in actual urine, the detection limit is 0.149mM, and the linear range of detection is 0-1.014 mM. The non-enzymatic fluorescence sensing method has good anti-interference performance on albumin, creatinine and cysteine in urine and good selectivity on glucose. The concentration of glucose in urine of normal healthy human body is 0.2-0.3 mM [ D.K. Sen, G.S. Sarin, Tear glucose-levels in normal blood lipids and in diabetes-properties, Brit. J. Ophthalmol 64(9) (1980) 693 + 695], so that the detection method can make quantitative and qualitative judgment on the condition of glucose metabolism disorder in human body.
Drawings
FIG. 1 is a schematic diagram of the preparation process of nitrogen-doped carbon quantum dots and the principle of detecting glucose;
FIG. 2 is an X-ray photoelectron spectrum of a nitrogen-doped carbon quantum dot in example 1;
FIG. 3 is a TEM image and size distribution of the nitrogen-doped carbon quantum dots in example 1;
FIG. 4 is an AFM picture of the nitrogen-doped carbon quantum dots of example 1;
FIG. 5 is an XRD pattern of the carbon doped nitrogen quantum dots of example 1;
FIG. 6 is a three-dimensional fluorescence spectrum of the N-doped carbon quantum dot in example 1;
FIG. 7 is a graph of the response of fluorescence spectra obtained from the measurement of human urine samples containing different concentrations of glucose in example 1, showing the relationship between the fluorescence intensity and the glucose concentration;
in the figure: a is a fluorescence spectrum response diagram of 0mM glucose; b is the fluorescence spectrum response diagram of 0.149mM glucose; c is the fluorescence spectrum response diagram of 0.297mM glucose; d is the fluorescence spectrum response diagram of 0.443mM glucose; e is the fluorescence spectrum response diagram of 0.558mM glucose; f is the fluorescence spectrum response diagram of 0.731mM glucose; g is the fluorescence spectrum response diagram of 0.873mM glucose; h is a fluorescence spectrum response diagram of 1.014mM glucose; i is the fluorescence spectrum response diagram of 1.153mM glucose; j is the fluorescence spectral response plot of 1.291mM glucose.
FIG. 8 is a graph showing the linear range of fluorescence intensity of the spots for detecting glucose at different concentrations in example 1;
FIG. 9 shows the fluorescence intensity of different solutions in the selective assay for glucose sensing with nitrogen-doped carbon quantum dots of example 1;
fig. 10 is a TEM picture of the nitrogen-doped carbon quantum dots in example 2.
Detailed Description
The invention relates to a non-enzymatic fluorescence sensing detection method for detecting glucose in human urine, which is based on the complexation of glucose and nitrogen atoms at the edge defect of a nitrogen-doped carbon quantum dot and ensures high selectivity to glucose. The nitrogen-doped carbon quantum dots have excellent fluorescence emission performance and play a role in signal amplification in the method; the nitrogen-doped carbon quantum dots have stable chemical properties, and the repeatability and stability of sensing detection are improved.
The invention is further described with reference to the following figures and examples.
Example 1
1. The nitrogen-doped carbon quantum dot is prepared by the following specific processes:
referring to fig. 1, the reactant formamide solvent is poured into a polytetrafluoroethylene container, the volume of the solvent is ensured to be 80% of the volume of the container, the reaction temperature is 180 ℃, the reaction time is 1.5 h, ammonia gas is released by thermal polymerization, and a mixed solution of nitrogen-doped carbon quantum dots and formamide is formed, namely the N-C QDs solution.
FIG. 2 is an X-ray photoelectron spectrum of the nitrogen-doped carbon quantum dot synthesized in this example, from which it can be seen that the main elements of the product are carbon and nitrogen, and the surface may adsorb a certain oxygen functional group; the atomic ratio of carbon to nitrogen is 0.29, which is less than that of carbon-nitrogen-four (g-C)3N4) The ratio of carbon to nitrogen atoms (0.75), so the product synthesized by the process should be nitrogen-doped carbon nanomaterial.
Fig. 3 is a TEM image and a size distribution of the nitrogen-doped carbon quantum dots synthesized in this example, and it can be seen that the morphology of the product is nitrogen-doped carbon quantum dots with an average size of 5 nm.
Fig. 4 is an AFM image of the synthesized nitrogen-doped carbon quantum dot in this example, and it can be seen that the thickness of the nitrogen-doped carbon quantum dot is 0.35nm, which is a single layer.
FIG. 5 is an XRD spectrum of the nitrogen-doped carbon quantum dot synthesized in this example, and it can be seen that the crystalStructural and graphitized carbon-nitrogen-carbon-nitrogen-four (g-C)3N4) The structures are the same, and the positions of diffraction peaks of (002) crystal faces of the two are the same, which shows that the nitrogen doping process is equivalent to that partial carbon atoms in the graphene are replaced by nitrogen atoms.
FIG. 6 is a three-dimensional fluorescence spectrum of the nitrogen-doped carbon quantum dot synthesized in this example, and it can be seen that the band gap of the nitrogen-doped carbon quantum dot is 3.0-3.5 eV, which illustrates the semiconductor characteristics of the carbon quantum dot caused by doping.
2. The non-enzymatic fluorescence sensing detection method for glucose in human urine based on the nitrogen-doped carbon quantum dots comprises the following steps:
(1) uniformly mixing the N-C QDs solution obtained in the above process with urine containing glucose with different concentrations to ensure that the volume of the N-C QDs solution is the same as that of the urine; under the excitation of incident light with the wavelength of 350nm, fluorescence with different intensities is generated, and the sensitivity and the linear range of the sensing detection method are obtained according to the relation between the concentration of glucose in urine and the fluorescence intensity;
(2) adding equal volume of the nitrogen-doped carbon quantum dot solution into the urine to be detected, uniformly mixing, detecting the fluorescence intensity under the excitation of incident light with the wavelength of 350nm, and calculating the content of the glucose in the urine to be detected according to the linear relation between the concentration of the glucose and the fluorescence intensity in the step (1).
FIG. 7 is a graph of the response of fluorescence spectrum obtained by detecting human urine samples containing different concentrations of glucose using the N-doped carbon quantum dots synthesized in this example, and it can be seen that the fluorescence intensity decreases with the increase of the glucose concentration.
FIG. 8 is a graph showing the linear range of the fluorescence intensity of the N-doped carbon quantum dots synthesized in this example, wherein the linear range of the fluorescence sensing detection method is 0-1.014 mM (R2 = 0.966), and the detection limit is 0.149 mM; the concentration of glucose in urine of a normal and healthy human body is 0.2-0.3 mM, so that the sensing detection method can make quantitative and qualitative judgment on the metabolic disturbance of glucose in the human body.
3. Selectivity test for glucose:
adding different interferents into the prepared N-C QDs solution for evaluating the selectivity of the sensing detection method; the interferents include urine, urine and cysteine (200. mu.M/L), urine, albumin (40 mg/mM) and creatinine (40 mg/mM), urine and glucose; their corresponding fluorescence intensities were tested at the same excitation wavelength.
Fig. 9 is a graph of a selectivity test of the nitrogen-doped carbon quantum dot synthesized in this embodiment on glucose, and it can be seen from the graph that fluorescence of the nitrogen-doped carbon quantum dot can be selectively quenched by glucose, and no significant fluorescence quenching phenomenon occurs on other interfering substances, which indicates that the sensing detection method has good anti-interference performance and selectivity.
The high selectivity of the detection method provided by the invention for glucose is derived from hydrogen bonds formed by dangling bonds at the edge of the nitrogen-doped carbon quantum dots and glucose molecules.
Example 2
1. The nitrogen-doped carbon quantum dot is prepared by the following specific processes:
pouring a reactant formamide solvent into a polytetrafluoroethylene container, ensuring that the volume of the solvent accounts for 80% of the volume of the container, the reaction temperature is 150 ℃, the reaction time is 12 hours, and thermally polymerizing to release ammonia gas to form a mixed solution of nitrogen-doped carbon quantum dots and formamide, namely the N-C QDs solution.
Fig. 10 is a TEM image of the synthesized nitrogen-doped carbon quantum dots of example 2, and it can be seen that the morphology of the product is the nitrogen-doped carbon quantum dots with an average size of 8 nm.
2. The non-enzymatic fluorescence sensing detection method for glucose in human urine based on the nitrogen-doped carbon quantum dots comprises the following steps:
(1) uniformly mixing the N-C QDs solution obtained in the process with urine containing glucose with different concentrations to ensure that the volume of the N-C QDs is the same as that of the urine, generating fluorescence with different intensities under the excitation of incident light with the wavelength of 350nm, and obtaining the sensitivity and the linear range of the sensing detection method according to the relationship between the concentration of the glucose in the urine and the fluorescence intensity;
(2) adding an equal volume of the nitrogen-doped carbon quantum dot solution into the urine to be detected, uniformly mixing, detecting the fluorescence intensity under the excitation of incident light with the wavelength of 350nm, and calculating the content of the glucose in the urine to be detected according to the linear relation between the concentration of the glucose and the fluorescence intensity in the step (1).
3. Selectivity test for glucose:
adding different interferents into the prepared N-C QDs solution for evaluating the selectivity of the sensing detection method; the interferents include urine, urine and cysteine (200. mu.M/L), urine, albumin (40 mg/mM) and creatinine (40 mg/mM), urine and glucose; their corresponding fluorescence intensities were tested at the same excitation wavelength.
Example 3
1. The nitrogen-doped carbon quantum dot is prepared by the following specific processes:
pouring a reactant formamide solvent into a polytetrafluoroethylene container, ensuring that the volume of the solvent accounts for 80% of the volume of the container, the reaction temperature is 200 ℃, the reaction time is 1h, and thermally polymerizing to release ammonia gas to form a mixed solution of nitrogen-doped carbon quantum dots and formamide, namely the N-C QDs solution.
The non-enzymatic fluorescence sensing detection method for glucose in human urine based on the nitrogen-doped carbon quantum dots and the selective test of glucose are the same as the steps of the embodiment 1 and the embodiment 2, and are not repeated.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Claims (5)
1. A non-enzymatic fluorescence sensing detection method for glucose in human urine is characterized in that: based on the fluorescence emission performance of the nitrogen-doped carbon quantum dots, the fluorescence performance of the nitrogen-doped carbon quantum dots is selectively quenched by glucose, so that qualitative and quantitative detection of the glucose in the urine is realized;
the precursor of the nitrogen-doped carbon quantum dot is formamide, so that a nitrogen-doped carbon quantum dot solution taking formamide as a solvent is formed; the preparation method of the nitrogen-doped carbon quantum dot solution comprises the following steps:
and pouring a formamide solvent into the reaction kettle, wherein the volume of the formamide solvent accounts for 60-80% of the volume of the container, the reaction temperature is 120-220 ℃, the reaction time is 0.5-20 h, ammonia gas is released by thermal polymerization to form a mixed solution of the nitrogen-doped carbon quantum dots and the formamide, and the mass concentration of the nitrogen-doped carbon quantum dots is 0.025-1% according to the difference of the time or temperature of the solvothermal reaction.
2. The method for non-enzymatic fluorescence sensing of glucose in human urine according to claim 1, wherein: the method comprises the following specific steps:
1) mixing urine of a healthy human body and the nitrogen-doped carbon quantum dot solution according to the volume ratio of 1:1, adding glucose solutions with different concentrations, and uniformly mixing to form samples to be detected containing glucose with different concentrations;
2) exciting the sample to be detected formed in the step 1) under incident light with the wavelength of 300-400 nm, taking the nitrogen-doped carbon quantum dots as fluorescent probes to generate fluorescence with different intensities, and obtaining the sensitivity and the linear range of the sensing detection method according to the relation between the concentration of glucose in urine and the fluorescence intensity;
3) for the detection of an actual sample, adding an equal volume of the nitrogen-doped carbon quantum dot solution into human urine to be detected, uniformly mixing, obtaining the fluorescence intensity of the sample to be detected by adopting the exciting light with the same wavelength in the step 2), and calculating the glucose content in the urine according to the linear relation between the concentration of glucose and the fluorescence intensity in the step 2).
3. The method for non-enzymatic fluorescence sensing of glucose in human urine according to claim 1, wherein: when the nitrogen-doped carbon quantum dot is prepared, the lower the reaction temperature is, the longer the reaction time is.
4. The method for non-enzymatic fluorescence sensing of glucose in human urine according to claim 1, wherein: the detection method has high selectivity on glucose and is derived from hydrogen bonds formed by dangling bonds at the edge of the nitrogen-doped carbon quantum dots and glucose molecules.
5. The method for non-enzymatic fluorescence sensing of glucose in human urine according to claim 1, wherein: the method for verifying the selective quenching of the fluorescence property of the nitrogen-doped carbon quantum dot by glucose is as follows,
the method comprises the steps of selecting four original solutions to be tested, namely urine, a mixed solution of urine and cysteine, a mixed solution of urine, albumin and creatinine, and a mixed solution of urine and glucose, adding nitrogen-doped carbon quantum dot solutions into the four original solutions to be tested respectively, and testing the fluorescence intensity of the four solutions to be tested under the excitation of light with the same wavelength.
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