CN110455753A - The non-enzymatic fluorescence sense detection method of glucose in a kind of human urine - Google Patents
The non-enzymatic fluorescence sense detection method of glucose in a kind of human urine Download PDFInfo
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Abstract
The invention discloses a kind of non-enzymatic fluorescence sense detection methods of glucose in human urine.Fluorescence probe used in the sensing detection method is Nitrogen doped carbon quantum dot.Nitrogen doped carbon quantum dot solution is obtained by the heat polymerization of formamide, and for solvent thermal reaction temperature at 120 ~ 220 DEG C, the reaction time is 0.5 ~ 20h, and thermal polymerization discharges ammonia；The Nitrogen doped carbon quantum dot solution for taking the above process to obtain is uniformly mixed with the urine containing different glucose respectively, guarantee that Nitrogen doped carbon quantum dot solution is identical with volume of urine, their fluorescent emission intensity is tested, excitation wavelength is 300 ~ 400nm, obtains detection limit and the range of linearity；Urine to be measured mixes in equal volume with Nitrogen doped carbon quantum dot solution, passes through the concentration of glucose known to fluorescence intensity.A kind of non-enzymatic fluorescence sense detection method provided by the invention can carry out quantitative detection to glucose, have good interference free performance and selection performance.
Description
Technical field
The present invention relates to a kind of non-enzymatic fluorescence sense detection methods of glucose in human urine, belong to analysis and detection technology
Field.
Background technique
Develop quick, accurate, highly selective glucose sensing approach, can be widely used for clinical diagnosis, diabetes mellitus prevention,
The fields such as Food Monitoring, drugs analysis and biochemical reaction process research.Currently, glucose sensing detection method is broadly divided into enzyme base
Or non-enzymatic based sensor.Enzyme base sensing detection method requires harshness to testing conditions, because used enzyme (such as glucose dehydrogenation
Enzyme and glucose oxidase) to the pH of environment, temperature, humidity quite sensitive, therefore the stability of enzyme base method for sensing is relatively 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 sensing inspection of non-enzymatic base
The stability of survey method has improvement substantially compared with enzyme base sensing method；The electrochemical sensing inspection that non-enzymatic method for sensing is mainly concentrated
It surveys, their testing principle is based on electrode material to the electrochemical oxidation of glucose, but high oxidation overpotential will lead to electrode
Material corrosion, formation intermediate product generate electrode fouling, above situation can all influence its accuracy.
Commercialized glucose sensor Technical comparing is mature, mainly includes two kinds of inspection policies: enzyme based electrochemical sensing
Device and the light sensing method scattered based on light.Other than the shortcomings that being previously mentioned enzyme based sensor, commercialized glucose sensor
The overwhelming majority needs to acquire blood sample, and needs to carry out cumbersome sample pretreatment process, therefore develops hurtless measure, non-enzymatic sensing
Detection method is significant.Fluorescence sense method is able to achieve human blood glucose, unlike electrochemical sensing detection method is vulnerable to electrolyte
Influence, therefore fluorescence sense method is a kind of ideal detection glucose sensing approach [M.S. Steiner, A.
Duerkop, O.S. Wolfbeis, Optical methods for sensing glucose, Chem. Soc. Rev.
40(9) (2011) 4805-4839]。
The performance (such as selectivity, sensitivity, stability) of fluorescence sense method depends on fluorescent material or fluorescence probe
Design.Fluorescence sense detects glucose, and there are two types of modes: being acted on based on glucose and probe molecule, leads to the fluorophor of modification
Be quenched；Being quenched based on probe material fluorescence itself enhances.Former mode is usually used in bio-sensing field, latter
Mode is chiefly used in chemical sensitisation field.Compared with bio-sensing detection method, based on probe material fluorescence enhancement itself or it is quenched
The stability of chemical sensitisation detection method is more preferable, and it is small to detect the influence of environment (such as pH) to sensing capabilities.This fluorescence chemical passes
Sense detection method be based primarily upon inorganic nano material, especially quantum dot, such as carbon quantum dot, have good bio-compatibility,
Chemical stability, good fluorescence radiation performance, fermentoid and other catalytic performances, are widely used in fluorescence sense field.Carbon
Quantum dot is also easy to be modified, for example can be further improved its fluorescence radiation performance by element doping.It is reported that nitrating
The fluorescent yield (φ) of four quantum dot of carbon quantum dot or three nitrogen of carbon reach 0.3 ~ 0.9 [J. Wu, S.W. Yang, J.P. Li,
et al., Electron injection of phosphorus 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].But above-mentioned fluorescent material be only used for human configuration buffer (such as
NaOH and PBS) in, seldom consider to apply in actual scene or environment, such as blood or urine.Compared to the grape in blood
It is relatively reliable to detect glucose in urine, while avoiding and generating intrusive injury to human body for sugar.
Summary of the invention
Purpose: in order to overcome the deficiencies in the prior art, the present invention provides a kind of grape detected in human urine
Sugared non-enzymatic fluorescence sense detection method；Solvent thermal process is utilized for the first time, is had good stability by the thermal polymerization formation of formamide
Nitrogen doped carbon quantum dot, fluorescence can be selectively quenched by glucose molecule, quantify to realize to glucose sugar in urine
Detection；Urine to be measured has practical prospect without carrying out pre-treatment.
Technical solution: in order to solve the above technical problems, the technical solution adopted by the present invention are as follows:
A kind of glucose non-enzymatic fluorescence sense detection method in human urine, the fluorescent emission based on Nitrogen doped carbon quantum dot
Can, the fluorescence property of Nitrogen doped carbon quantum dot by glucose selective quenches, realize in urine glucose it is qualitative and quantitative
Detection.
Preferably, the glucose non-enzymatic fluorescence sense detection method in a kind of human urine, comprises the following specific steps that:
1) by the urine of healthy human body and Nitrogen doped carbon quantum dot solution using formamide as solvent after 1:1 is mixed by volume,
The glucose solution of various concentration is added, is uniformly mixed and forms the prepare liquid containing different glucose；
2) by the prepare liquid formed in step 1) under the incident light excitation that wavelength is 300 ~ 400nm, Nitrogen doped carbon quantum dot conduct
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 detected, mixes
After closing uniformly, using the exciting light of phase co-wavelength in step 2, the fluorescence intensity to test sample is obtained, according to glucose in step 2
Concentration and fluorescence intensity linear relationship calculate urine in glucose content.
Preferably, the presoma of the Nitrogen doped carbon quantum dot is formamide, is formed using formamide as the nitrating carbon amounts of solvent
Son point solution.
Preferably, the preparation method of the Nitrogen doped carbon quantum dot solution is as follows,
Raw material formamide solvent to be poured into polytetrafluoroethylcontainer container, the volume of formamide solvent accounts for the 60-80% of vessel volume,
For reaction temperature at 120 ~ 220 DEG C, the reaction time is 0.5 ~ 20h, and thermal polymerization discharges ammonia, forms Nitrogen doped carbon quantum dot and formamide
Mixed solution, 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%.
Preferably, when preparing Nitrogen doped carbon quantum dot, reaction temperature is lower, and the reaction time is longer.
Preferably, the detection method has glucose highly selective, is derived from Nitrogen doped carbon quantum dot edge dangling bond and Portugal
The hydrogen bond that grape glycan molecule is formed.
Preferably, 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.
The utility model has the advantages that the present invention utilizes the water-soluble good semiconductor Nitrogen doped carbon quantum dot of hydro-thermal method one-step synthesis, preparation
Method is simple, reliable.Nitrogen doped carbon quantum dot has good fluorescent emission performance, and its fluorescence can be selected by glucose molecule
Selecting property it is quenched.Based on the characteristic, it is applied to the quantitative detection of glucose in practical urine, detection is limited to 0.149 mM,
The range of linearity of detection is 0 ~ 1.014mM.The non-enzymatic fluorescence sense method is to albumin, kreatinin, the cysteine in urine
There is good selectivity with good interference free performance and to glucose.Concentration of glucose in the urine of Normal healthy individuals
For 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], therefore the detection side
Method can make judgement qualitatively and quantitatively to glucose metabolism disorders situation in human body.
Detailed description of the invention
Fig. 1 is the preparation process of Nitrogen doped carbon quantum dot and its schematic illustration for detecting glucose；
Fig. 2 is the x-ray photoelectron spectroscopy of Nitrogen doped carbon quantum dot in embodiment 1；
Fig. 3 is the TEM picture of Nitrogen doped carbon quantum dot and size distribution in embodiment 1；
Fig. 4 is the AFM picture of Nitrogen doped carbon quantum dot in embodiment 1；
Fig. 5 is the XRD spectrum 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 that fluorescence spectrum response diagram obtained by the human urine sample of the glucose containing various concentration is detected in embodiment 1, is embodied
The relationship of fluorescence intensity and concentration of glucose；
In figure: a is the fluorescence spectrum response diagram of 0mM glucose；B is the fluorescence spectrum response diagram of 0.149 mM 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 0.558mM grape
The fluorescence spectrum response diagram of sugar；F is the fluorescence spectrum response diagram of 0.731mM glucose；G is the fluorescence light of 0.873mM glucose
Compose response diagram；H is the fluorescence spectrum response diagram of 1.014mM glucose；I is the fluorescence spectrum response diagram of 1.153mM glucose；j
For the fluorescence spectrum response diagram of 1.291mM glucose.
Fig. 8 is that Nitrogen doped carbon quantum dot detects the glucose of various concentration and the range of linearity of its fluorescence intensity in embodiment 1；
Fig. 9 is the fluorescence intensity of different solutions in the selectivity test of Nitrogen doped carbon quantum dot sensing detection glucose in embodiment 1；
Figure 10 is the TEM picture of Nitrogen doped carbon quantum dot in embodiment 2.
Specific embodiment
The present invention relates to the glucose non-enzymatic fluorescence sense detection method in a kind of detection human urine, this method is based on Portugal
The complexing of nitrogen-atoms at grape sugar and Nitrogen doped carbon quantum dot edge defect guarantees to the highly selective of glucose.Nitrating carbon
Quantum dot has excellent fluorescent emission performance, plays signal amplification in the method；Nitrogen doped carbon quantum dot chemical property is steady
It is fixed, improve the reproducibility and stability of sensing detection.
The present invention will be further explained with reference to the accompanying drawings and examples.
Embodiment 1
1, Nitrogen doped carbon quantum dot is prepared, detailed process is as follows:
Refering to fig. 1, reactant formamide solvent is poured into polytetrafluoroethylcontainer container, guarantees that solvent volume accounts for the 80 of vessel volume
%, for reaction temperature at 180 DEG C, the reaction time is 1.5 h, and thermal polymerization discharges ammonia, forms Nitrogen doped carbon quantum dot and formamide
Mixed solution, as N-C QDs solution.
Fig. 2 is the x-ray photoelectron spectroscopy of the Nitrogen doped carbon quantum dot of embodiment synthesis, from figure it is found that product is mainly first
Element is carbon and nitrogen, and surface may adsorb certain oxygen functional group；The atomic ratio of carbon and nitrogen is 0.29, is less than three nitrogen of carbon, four (g-
C3N4) carbon and nitrogen-atoms ratio (0.75), therefore product synthesized by this process should be nitrating carbon nanomaterial.
Fig. 3 is TEM picture and the size distribution of the Nitrogen doped carbon quantum dot of embodiment synthesis, as seen from the figure, the shape of product
Looks are the Nitrogen doped carbon quantum dot that average-size is 5nm.
Fig. 4 is the AFM picture of the Nitrogen doped carbon quantum dot of embodiment synthesis, as seen from the figure, the thickness of Nitrogen doped carbon quantum dot
For 0.35nm, as single layer.
Fig. 5 is the XRD spectrum of the Nitrogen doped carbon quantum dot of embodiment synthesis, as seen from the figure, the crystal structure and graphitization
Three nitrogen of carbon, four (g-C3N4) structure is identical, (002) crystallographic plane diffraction peak position of the two is identical, illustrates that N doping process is equal to
Part carbon atom in graphene is replaced by nitrogen-atoms.
Fig. 6 is the three-dimensional fluorescence spectrum of the Nitrogen doped carbon quantum dot of embodiment synthesis, as seen from the figure, Nitrogen doped carbon quantum dot
Band gap is 3.0 ~ 3.5eV, illustrates that doping results in the characteristic of semiconductor of carbon quantum dot.
2, glucose non-enzymatic fluorescence sense detection method in the human urine based on Nitrogen doped carbon quantum dot, including walk as follows
It is rapid:
(1) the N-C QDs solution for taking the above process to obtain is uniformly mixed with the urine containing different glucose, guarantees N-C
QDs solution is identical with volume of urine；In the case where wavelength is the excitation of 350nm incident light, the fluorescence of varying strength is generated, according in urine
The concentration of glucose and the relationship of fluorescence intensity, obtain the sensitivity and linear measurement range of the sensing detection method；
(2) isometric Nitrogen doped carbon quantum dot solution is added in urine to be detected, after mixing, is equally in wavelength
Under the incident light excitation of 350nm, fluorescence intensity, according to the concentration of glucose and the line of fluorescence intensity in above-mentioned steps (1)
Sexual intercourse calculates the content of glucose in urine to be detected.
Fig. 7 is that the Nitrogen doped carbon quantum dot synthesized using the embodiment detects the human urine sample of the glucose containing various concentration
Fluorescence spectrum response diagram obtained by product, as seen from the figure, with the increase of concentration of glucose, fluorescence intensity is reduced.
The glucose for the Nitrogen doped carbon quantum dot detection various concentration that Fig. 8 synthesizes for the embodiment is linear with its fluorescence intensity
Areal map, as seen from the figure, the range of linearity of the fluorescence sense detection method are 0 ~ 1.014mM(R2=0.966), detection is limited to
0.149mM；Concentration of glucose in the urine of Normal healthy individuals is 0.2 ~ 0.3mM, therefore the sensing detection method can be to human body
Glucose metabolism disorders make judgement qualitatively and quantitatively.
3, the selectivity test of glucose:
Take the N-C QDs solution of aforementioned preparation that disturbance object is added, for assessing the selectivity of the sensing detection method；It is described
Chaff interferent includes urine, urine and cysteine (200 μM/L), urine, albumin (40mg/mM) and kreatinin (40mg/mM),
Urine and glucose；The fluorescence intensity corresponding to them is tested under identical excitation wavelength.
Fig. 9 is selectivity test figure of the Nitrogen doped carbon quantum dot to glucose of embodiment synthesis, as seen from the figure, nitrating carbon
The fluorescence of quantum dot can be quenched selectively by glucose, and there is no significant Fluorescence-quenchings to produce to other interfering substances
It is raw, illustrate that the sensing detection method has good interference free performance and selection performance.
Detection method provided by the invention is derived from Nitrogen doped carbon quantum dot edge dangling bond and grape to the highly selective of glucose
The hydrogen bond that glycan molecule is formed.
Embodiment 2
1, Nitrogen doped carbon quantum dot is prepared, detailed process is as follows:
Reactant formamide solvent is poured into polytetrafluoroethylcontainer container, guarantees that solvent volume accounts for 80 % of vessel volume, is reacted
For temperature at 150 DEG C, reaction time 12h, thermal polymerization discharges ammonia, forms the mixed solution of Nitrogen doped carbon quantum dot and formamide,
As N-C QDs solution.
Figure 10 is the TEM picture for the Nitrogen doped carbon quantum dot that embodiment 2 synthesizes, and as seen from the figure, the pattern of product is average ruler
The very little Nitrogen doped carbon quantum dot for 8nm.
2, glucose non-enzymatic fluorescence sense detection method in the human urine based on Nitrogen doped carbon quantum dot, including walk as follows
It is rapid:
(1) the N-C QDs solution for taking the above process to obtain is uniformly mixed with the urine containing different glucose, guarantees N-C
QDs is identical with volume of urine, in the case where wavelength is the excitation of 350nm incident light, the fluorescence of varying strength is generated, according to grape in urine
The concentration of sugar and the relationship of fluorescence intensity, obtain the sensitivity and linear measurement range of the sensing detection method；
(2) isometric Nitrogen doped carbon quantum dot solution is added in urine to be detected, is 350nm in wavelength after mixing
Incident light excitation under, fluorescence intensity, according to the concentration of glucose and the linear relationship of fluorescence intensity in above-mentioned steps (1)
Calculate the content of glucose in urine to be measured.
3, the selectivity test of glucose:
Take the N-C QDs solution of aforementioned preparation that disturbance object is added, for assessing the selectivity of the sensing detection method；It is described
Chaff interferent includes urine, urine and cysteine (200 μM/L), urine, albumin (40mg/mM) and kreatinin (40mg/mM),
Urine and glucose；The fluorescence intensity corresponding to them is tested under identical excitation wavelength.
Embodiment 3
1, Nitrogen doped carbon quantum dot is prepared, detailed process is as follows:
Reactant formamide solvent is poured into polytetrafluoroethylcontainer container, guarantees that solvent volume accounts for 80 % of vessel volume, is reacted
For temperature at 200 DEG C, reaction time 1h, thermal polymerization discharges ammonia, forms the mixed solution of Nitrogen doped carbon quantum dot and formamide, i.e.,
For N-C QDs solution.
The choosing of glucose non-enzymatic fluorescence sense detection method and glucose in human urine based on Nitrogen doped carbon quantum dot
The test of selecting property is identical as embodiment 1,2 step of embodiment, repeats no more.
The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as 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.
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