CN110455753A - A non-enzymatic fluorescent sensing method for glucose in human urine - Google Patents

Abstract

The invention discloses a non-enzyme fluorescent sensing detection method for glucose in human urine. The fluorescent probe used in 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 solvothermal reaction temperature is 120~220°C, the reaction time is 0.5~20h, and ammonia gas is released by thermal polymerization; the nitrogen-doped carbon quantum dot solution obtained by the above process is taken Mix well with urine containing different concentrations of glucose, ensure that the nitrogen-doped carbon quantum dot solution and urine have the same volume, test their fluorescence emission intensity, the excitation wavelength is 300~400nm, and obtain the detection limit and linear range; the urine to be tested It is mixed with nitrogen-doped carbon quantum dot solution in equal volume, and the concentration of glucose can be known by detecting the fluorescence intensity. The non-enzyme fluorescent sensing and detection method provided by the invention can quantitatively detect glucose and has good anti-interference performance and selectivity.

Description

A non-enzymatic fluorescent sensing method for glucose in human urine

technical field

The invention relates to a non-enzyme fluorescent sensing detection method for glucose in human urine, belonging to the technical field of analysis and detection.

Background technique

Develop fast, accurate and highly selective glucose detection methods, which can be widely used in clinical diagnosis, diabetes prevention, food monitoring, drug analysis and biochemical reaction process research and other fields. Currently, glucose sensing methods are mainly classified into enzyme-based or non-enzyme-based sensors. Enzyme-based sensing detection methods require harsh detection conditions, because the enzymes used (such as glucose dehydrogenase and glucose oxidase) are extremely sensitive to the pH, temperature, and humidity of the environment, so the stability of enzyme-based sensing methods is relatively low. Poor [H. Zhu, L. Li, W. Zhou et al., Advances in non-enzymatic glucose sensors based on metal oxides, J. Mater. Chem. B 4(46) (2016) 7333-7349]. The stability of non-enzyme-based sensing detection methods is substantially improved compared with enzyme-based sensing methods; non-enzyme-based sensing methods mainly focus on electrochemical sensing detection, and their detection principle is based on the electrochemical oxidation of glucose by electrode materials. However, a high oxidation overpotential will lead to corrosion of electrode materials and electrode contamination by intermediate products formed, all of which will affect its accuracy.

The commercial glucose sensor technology is relatively mature, mainly including two detection strategies: enzyme-based electrochemical sensor and light-scattering-based light sensing method. In addition to the shortcomings of enzyme-based sensors mentioned above, most commercial glucose sensors require blood samples and tedious sample pretreatment processes, so the development of non-invasive, non-enzyme sensing and detection methods is of great significance. The fluorescent sensing method can realize non-invasive detection, unlike the electrochemical sensing detection method which is easily affected by electrolytes, so the fluorescent 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 (eg, selectivity, sensitivity, stability) of fluorescent sensing methods depends on the design of fluorescent materials or fluorescent probes. There are two modes for fluorescent sensing to detect glucose: based on the interaction between glucose and the probe molecule, resulting in the quenching of the modified fluorescent group; based on the quenching or enhancement of the fluorescence of the probe material itself. The former mode is often used in the field of biosensing, and the latter mode is mostly used in the field of chemical sensing. Compared with biosensing assays, chemical sensing assays based on fluorescence enhancement or quenching of the probe material itself have better stability, and the detection environment (such as pH) has little impact on sensing performance. This fluorescent chemical sensing method is mainly based on inorganic nanomaterials, especially quantum dots, such as carbon quantum dots, which have good biocompatibility, chemical stability, good fluorescence properties, enzyme-like properties and other catalytic properties. Widely used in the field of fluorescence sensing. Carbon quantum dots are also easy to modify, for example, element doping can further improve their fluorescence properties. It is reported that the fluorescence yield (φ) of nitrogen-doped carbon quantum dots or carbon three nitrogen four quantum dots reaches 0.3~0.9 [J. Wu, SW Yang, JP Li, et al., Electron injection of phosphorus doped gC ₃ N ₄ quantum dots:controllable photoluminescence emission wavelength in the whole visible lightrange with high quantum yield, Adv. Opt. Mater. 4(12) (2016) 2095-2101; MCRong, ZX Cai, L. Xie, et al., Study on the ultrahigh quantum yield fluorescent P, OgC ₃ N ₄ nanodots and its application in cell imaging, Chem. Eur. J. 22(27) (2016) 9387-9395]. However, the above-mentioned fluorescent materials are only used in artificially configured buffers (such as NaOH and PBS), and little consideration is given to practical scenarios or environmental applications, such as blood or urine. Compared with glucose in blood, the detection of glucose in urine is more reliable, while avoiding invasive damage to the human body.

Contents of the invention

Purpose: In order to overcome the deficiencies in the prior art, the present invention provides a non-enzymatic fluorescent sensing method for detecting glucose in human urine; for the first time, a solvothermal method is used to form a stable doped substance through thermal polymerization of formamide. Nitrogen-carbon quantum dots, whose fluorescence can be selectively quenched by glucose molecules, thereby realizing the quantitative detection of glucose in urine; the urine to be tested does not need to be pre-treated, and has practical prospects.

Technical solution: In order to solve the above-mentioned technical problems, the technical solution adopted in the present invention is:

A non-enzymatic fluorescent sensing method for glucose in human urine, based on the fluorescence emission properties of nitrogen-doped carbon quantum dots, the fluorescence properties of nitrogen-doped carbon quantum dots are selectively quenched by glucose, realizing the detection of glucose in urine Qualitative and quantitative testing.

Preferably, a non-enzymatic fluorescent sensing method for glucose in human urine comprises the following specific steps:

1) After mixing the urine of a healthy human body and the nitrogen-doped carbon quantum dot solution with formamide as a solvent at a volume ratio of 1:1, add glucose solutions of different concentrations, and mix well to form a test solution containing different concentrations of glucose;

2) The liquid to be tested formed in step 1) is excited by incident light with a wavelength of 300-400nm, and nitrogen-doped carbon quantum dots are used as fluorescent probes to produce different intensities of fluorescence, according to the concentration and fluorescence intensity of glucose in the urine The relationship of obtaining the sensitivity and the linear range of the sensing detection method;

3) For the detection of actual samples, add an equal volume of nitrogen-doped carbon quantum dot solution to the human urine to be tested, and after mixing evenly, use the excitation light of the same wavelength in step 2) to obtain the fluorescence intensity of the sample to be tested. Calculate the glucose content in the urine according to the linear relationship between the concentration of glucose and the fluorescence intensity in step 2).

Preferably, the precursor of the nitrogen-doped carbon quantum dots is formamide, forming a nitrogen-doped carbon quantum dot solution using formamide as a solvent.

Preferably, the preparation method of the nitrogen-doped carbon quantum dot solution is as follows,

Pour the raw formamide solvent into a polytetrafluoroethylene container, the volume of the formamide solvent accounts for 60-80% of the container volume, the reaction temperature is 120-220°C, the reaction time is 0.5-20h, thermal polymerization releases ammonia gas, and forms For the mixed solution of nitrogen-doped carbon quantum dots and formamide, the mass concentration of nitrogen-doped carbon quantum dots is 0.025-1% according to the time or temperature of the solvothermal reaction.

Preferably, when preparing nitrogen-doped carbon quantum dots, the lower the reaction temperature, the longer the reaction time.

Preferably, the detection method has high selectivity for glucose, which originates from the hydrogen bond formed by the edge dangling bonds of nitrogen-doped carbon quantum dots and glucose molecules.

Preferably, the method for verifying the selective quenching of the fluorescence properties of nitrogen-doped carbon quantum dots by glucose is as follows,

Select five original liquids to be tested, namely urine, mixed liquid of urine and cysteine, mixed liquid of urine, albumin and creatinine, mixed liquid of urine and glucose, in the five original liquids to be tested respectively The nitrogen-doped carbon quantum dot solution was added, and the fluorescence luminescence intensity of the five liquids to be tested was tested under the same wavelength of light excitation.

Beneficial effects: the invention uses a hydrothermal method to synthesize semiconductor nitrogen-doped carbon quantum dots with good water solubility in one step, and the preparation method is simple and reliable. Nitrogen-doped carbon quantum dots have good fluorescence emission properties, and its fluorescence can be selectively quenched by glucose molecules. Based on this characteristic, it was applied to the quantitative detection of glucose in actual urine, with a detection limit of 0.149 mM and a linear range of 0-1.014 mM. The non-enzymatic fluorescent sensing method has good anti-interference performance for albumin, creatinine and cysteine in urine and good selectivity for glucose. The glucose concentration in the urine of normal healthy people is 0.2~0.3mM [D. K. Sen, G. S. Sarin, Tear glucose-levels in normal people and in diabetic-patients, Brit. J. Ophthalmol 64(9) (1980) 693-695] , so the detection method can make quantitative and qualitative judgments on the glucose metabolism disorder in the human body.

Description of drawings

1 is a schematic diagram of the preparation process of nitrogen-doped carbon quantum dots and the principle of detecting glucose;

Fig. 2 is the X-ray photoelectron spectrum of nitrogen-doped carbon quantum dot in embodiment 1;

Fig. 3 is the TEM picture and size distribution of nitrogen-doped carbon quantum dot in embodiment 1;

Fig. 4 is the AFM picture of nitrogen-doped carbon quantum dot in embodiment 1;

Fig. 5 is the XRD collection of illustrative plates of nitrogen-doped carbon quantum dot in embodiment 1;

Fig. 6 is the three-dimensional fluorescence spectrum of nitrogen-doped carbon quantum dot in embodiment 1;

Fig. 7 is the fluorescence spectrum response graph obtained by detecting human urine samples containing different concentrations of glucose in Example 1, reflecting the relationship between fluorescence intensity and glucose concentration;

In the figure: a is the fluorescence spectral response diagram of 0mM glucose; b is the fluorescence spectral response diagram of 0.149 mM glucose; c is the fluorescence spectral response diagram of 0.297mM glucose; d is the fluorescence spectral response diagram of 0.443mM glucose; e is 0.558mM The fluorescence spectral response diagram of glucose; f is the fluorescence spectral response diagram of 0.731mM glucose; g is the fluorescence spectral response diagram of 0.873mM glucose; h is the fluorescence spectral response diagram of 1.014mM glucose; i is the fluorescence spectral response diagram of 1.153mM glucose ; j is the fluorescence spectral response graph of 1.291 mM glucose.

Fig. 8 is the linear range of different concentrations of glucose and its fluorescence intensity detected by nitrogen-doped carbon quantum dots in Example 1;

Fig. 9 is the fluorescence intensity of different solutions in the selectivity test of nitrogen-doped carbon quantum dot sensor detection glucose in embodiment 1;

10 is a TEM picture of nitrogen-doped carbon quantum dots in Example 2.

Detailed ways

The invention relates to a non-enzyme fluorescent sensing detection method for detecting glucose in human urine. The method is based on the complexation of glucose with nitrogen atoms at edge defects of nitrogen-doped carbon quantum dots, thereby ensuring high selectivity for glucose. The nitrogen-doped carbon quantum dot has excellent fluorescence emission performance, and plays a role in signal amplification in this method; the nitrogen-doped carbon quantum dot has stable chemical properties, and improves the reproducibility and stability of sensing and detection.

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Example 1

1. Preparation of nitrogen-doped carbon quantum dots, the specific process is as follows:

Referring to Figure 1, pour the reactant formamide solvent into a polytetrafluoroethylene container to ensure that the volume of the solvent accounts for 80% of the container volume, the reaction temperature is 180 °C, and the reaction time is 1.5 h. Thermal polymerization releases ammonia gas and forms nitrogen-doped The mixed solution of carbon quantum dots and formamide is the N-C QDs solution.

Figure 2 is the X-ray photoelectron spectrum of the nitrogen-doped carbon quantum dots synthesized in this embodiment, as can be seen from the figure, the main elements of the product are carbon and nitrogen, and certain oxygen functional groups may be adsorbed on the surface; the atomic ratio of carbon and nitrogen is 0.29, which is less than that of carbon The carbon to nitrogen atomic ratio of trinitrogen tetra(gC ₃ N ₄ ) is 0.75, so the product synthesized in this process should be a nitrogen-doped carbon nanomaterial.

Fig. 3 is the TEM picture and size distribution of the nitrogen-doped carbon quantum dots synthesized in this embodiment. It can be seen from the figure that the morphology of the product is nitrogen-doped carbon quantum dots with an average size of 5 nm.

Fig. 4 is an AFM picture of the nitrogen-doped carbon quantum dots synthesized in this embodiment. It can be seen from the figure that the nitrogen-doped carbon quantum dots have a thickness of 0.35 nm, which is a single layer.

Figure 5 is the XRD spectrum of the nitrogen-doped carbon quantum dots synthesized in this example. It can be seen from the figure that the crystal structure is the same as that of graphitized carbon three nitrogen four (gC ₃ N ₄ ) structure, and the (002) crystal plane diffraction of the two The peak positions are the same, indicating that the nitrogen doping process is equivalent to the replacement of some carbon atoms in graphene by nitrogen atoms.

Figure 6 is the three-dimensional fluorescence spectrum of the nitrogen-doped carbon quantum dots synthesized in this example. It can be seen from the figure that the band gap of the nitrogen-doped carbon quantum dots is 3.0-3.5eV, indicating that doping leads to the semiconductor properties of the carbon quantum dots.

2. A non-enzymatic fluorescent sensing method for glucose in human urine based on nitrogen-doped carbon quantum dots, comprising the following steps:

(1) Take the N-C QDs solution obtained in the above process and mix it with urine containing different concentrations of glucose to ensure that the volume of the N-CQDs solution and urine are the same; under the excitation of incident light with a wavelength of 350nm, different intensities of fluorescence are produced. The relationship between the concentration of glucose in the solution and the fluorescence intensity was used to obtain the sensitivity and linear range of the sensing method;

(2) Add an equal volume of nitrogen-doped carbon quantum dot solution to the urine to be tested. After mixing evenly, the fluorescence intensity is also detected under the excitation of incident light with a wavelength of 350nm. According to the concentration of glucose in the above step (1), The linear relationship between the fluorescence intensity and the fluorescence intensity is used to calculate the glucose content in the urine to be detected.

Fig. 7 is a fluorescence spectral response diagram obtained by using the nitrogen-doped carbon quantum dots synthesized in this embodiment to detect human urine samples containing different concentrations of glucose. It can be seen from the figure that the fluorescence intensity decreases with the increase of the glucose concentration.

Figure 8 is a linear range diagram of the nitrogen-doped carbon quantum dots synthesized in this example to detect different concentrations of glucose and its fluorescence intensity. It can be seen from the figure that the linear range of the fluorescence sensing detection method is 0~1.014mM (R2=0.966), The detection limit is 0.149mM; the glucose concentration in the urine of normal healthy people is 0.2~0.3mM, so the sensor detection method can make quantitative and qualitative judgments on the glucose metabolism disorder in the human body.

3. Glucose selectivity test:

Take the previously prepared N-C QDs solution and add different interfering substances to evaluate the selectivity of the sensing method; the interfering substances include urine, urine and cysteine (200 μM/L), urine, albumin (40mg/mM) and creatinine (40mg/mM), urine and glucose; test their corresponding fluorescence intensity at the same excitation wavelength.

Fig. 9 is the selectivity test diagram of the nitrogen-doped carbon quantum dots synthesized in this embodiment to glucose. It can be seen from the figure that the fluorescence of nitrogen-doped carbon quantum dots can be selectively quenched by glucose, but there is no significant effect on other interfering substances. The phenomenon of fluorescence quenching occurred, indicating that the sensing and detection method has good anti-interference performance and selectivity.

The high selectivity of the detection method provided by the invention for glucose originates from the hydrogen bond formed by the edge dangling bonds of nitrogen-doped carbon quantum dots and glucose molecules.

Example 2

1. Preparation of nitrogen-doped carbon quantum dots, the specific process is as follows:

Pour the reactant formamide solvent into a polytetrafluoroethylene container to ensure that the volume of the solvent accounts for 80% of the container volume, the reaction temperature is 150 ° C, and the reaction time is 12 hours. Thermal polymerization releases ammonia gas to form nitrogen-doped carbon quantum dots and formazan. The mixed solution of amides is the N-C QDs solution.

FIG. 10 is a TEM picture of the nitrogen-doped carbon quantum dots synthesized in Example 2. It can be seen from the figure that the morphology of the product is nitrogen-doped carbon quantum dots with an average size of 8 nm.

2. A non-enzymatic fluorescent sensing method for glucose in human urine based on nitrogen-doped carbon quantum dots, comprising the following steps:

(1) Take the N-C QDs solution obtained in the above process and mix it evenly with urine containing different concentrations of glucose to ensure that the volume of N-CQDs and urine is the same. Under the excitation of incident light with a wavelength of 350nm, different intensities of fluorescence will be produced. According to the urine The relationship between the concentration of glucose and the fluorescence intensity in the medium is obtained to obtain the sensitivity and linear range of the sensing detection method;

(2) Add an equal volume of nitrogen-doped carbon quantum dot solution to the urine to be tested. After mixing evenly, detect the fluorescence intensity under the excitation of incident light with a wavelength of 350nm. According to the concentration of glucose and The linear relationship of fluorescence intensity calculates the content of glucose in the urine to be tested.

3. Glucose selectivity test:

Take the previously prepared N-C QDs solution and add different interfering substances to evaluate the selectivity of the sensing method; the interfering substances include urine, urine and cysteine (200 μM/L), urine, albumin (40mg/mM) and creatinine (40mg/mM), urine and glucose; test their corresponding fluorescence intensity at the same excitation wavelength.

Example 3

1. Preparation of nitrogen-doped carbon quantum dots, the specific process is as follows:

Pour the reactant formamide solvent into a polytetrafluoroethylene container to ensure that the solvent volume accounts for 80% of the container volume, the reaction temperature is 200 ° C, and the reaction time is 1 h. Thermal polymerization releases ammonia gas to form nitrogen-doped carbon quantum dots and formazan. The mixed solution of amides is the N-C QDs solution.

The non-enzymatic fluorescent sensing and detection method of glucose in human urine based on nitrogen-doped carbon quantum dots, and the selective test of glucose are the same as those in Embodiment 1 and Embodiment 2, and will not be repeated here.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also possible. It should be regarded as the protection scope of the present invention.

Claims (7)

1. the non-enzymatic fluorescence sense detection method of glucose in a kind of human urine, it is characterised in that: be based on Nitrogen doped carbon quantum dot Fluorescent emission performance, the fluorescence property of Nitrogen doped carbon quantum dot by glucose selective quenches, and realizes to glucose in urine Qualitative and quantitative detection.

2. the non-enzymatic fluorescence sense detection method of glucose, feature exist in a kind of human urine according to claim 1 In: it comprises the following specific steps that:

1) by after the 1:1 mixing by volume of the urine of healthy human body and Nitrogen doped carbon quantum dot solution, the grape of various concentration is added Sugar juice, be uniformly mixed formed containing different glucose to test sample；

2) the Nitrogen doped carbon quantum dot conduct to test sample under the incident light excitation that wavelength is 300 ~ 400nm will be formed in step 1) Fluorescence probe generates the fluorescence of varying strength, according to the relationship of the concentration of glucose in urine and fluorescence intensity, obtains the sensing The sensitivity and linear measurement range of detection method；

3) for the detection of actual sample, isometric Nitrogen doped carbon quantum dot solution is added in human urine to be measured, mixing is equal After even, using the exciting light of phase co-wavelength in step 2, obtain the fluorescence intensity to test sample, according in step 2 glucose it is dense The linear relationship of degree and fluorescence intensity calculates the glucose content in urine.

3. the non-enzymatic fluorescence sense detection method of glucose, feature exist in a kind of human urine according to claim 1 In: the presoma of the Nitrogen doped carbon quantum dot is formamide, is formed using formamide as the Nitrogen doped carbon quantum dot solution of solvent.

4. the non-enzymatic fluorescence sense detection method of glucose, feature exist in a kind of human urine according to claim 3 In: the preparation method of the Nitrogen doped carbon quantum dot solution is as follows,

Formamide solvent is poured into reaction kettle, the volume of formamide solvent accounts for the 60-80% of vessel volume, and reaction temperature is 120 ~ 220 DEG C, the reaction time is 0.5 ~ 20h, and thermal polymerization discharges ammonia, forms the mixed solution of Nitrogen doped carbon quantum dot and formamide, root According to the time of solvent thermal reaction or the difference of temperature, the mass concentration of Nitrogen doped carbon quantum dot is 0.025 ~ 1%.

5. the non-enzymatic fluorescence sense detection method of glucose, feature exist in a kind of human urine according to claim 4 In: when preparation Nitrogen doped carbon quantum dot, reaction temperature is lower, and the reaction time is longer.

6. the non-enzymatic fluorescence sense detection method of glucose, feature exist in a kind of human urine according to claim 1 Have in: the detection method to glucose highly selective, is formed derived from Nitrogen doped carbon quantum dot edge dangling bond with glucose molecule Hydrogen bond.

7. the non-enzymatic fluorescence sense detection method of glucose, feature exist in a kind of human urine according to claim 1 In: glucose is as follows to the verification method of the selectivity quenching of Nitrogen doped carbon quantum dot fluorescence property,

Five kinds of original prepare liquids are selected, are urine, urine and cysteine mixed liquor respectively, urine, albumin and kreatinin are mixed Liquid, urine and glucose mixed liquor are closed, Nitrogen doped carbon quantum dot solution is separately added into five kinds of original prepare liquids, in phase co-wavelength The fluorescence radiation intensity of light excitation five kinds of prepare liquids of lower test.