CN108562564A - A kind of carbon quantum dot and preparation method and application for the detection of dextrase activity - Google Patents
A kind of carbon quantum dot and preparation method and application for the detection of dextrase activity Download PDFInfo
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The invention discloses a kind of carbon quantum dots and preparation method and application for the detection of dextrase activity.Using synanthrin and citric acid mixed solution as carbon source, by hydro-thermal method one-step synthesis green fluorescence carbon quantum dot, there is 520 nanometers most strong transmitting under 450 nanometers of excitations, and fluorescence emission wavelengths do not change with the change of excitation wavelength.It is probe using the carbon quantum dot, the fluorescence of carbon quantum dot increases with dextrase activity and quenched, and fluorescence intensity decreasing value is in good linear relationship with dextrase activity, constructs a kind of method of dextrase activity detection.It is novel that synanthrin and citric acid disclosed by the invention prepare carbon quantum dot selection, the dextrase activity detection method of structure has the characteristics that easy to operate, high sensitivity, selectivity are strong, it can be used for the quick detection of dextrase activity, the present invention fills up dextrase activity determination technological gap, has applications well foreground.
Description
Technical field
The invention belongs to the crossing domains of chemistry, biology and materialogy, are related to material preparation, the detection of biological enzyme activity,
More particularly to a kind of carbon quantum dot and preparation method and application for the detection of dextrase activity.
Background technology
Carbon quantum dot, typically refer to it is a kind of using carbon as essential element, size between 2~10nm, three dimensions all locate
It is a kind of nano-particle possessing special optical, electricity, biological characteristics in nano level crystal.With traditional semiconductor amount
Son point is compared, and while having excellent optical property as the carbon quantum dot that carbon source synthesizes using citric acid, is also had good water-soluble
Property and biocompatibility, this makes acquisition extensive use of the carbon quantum dot in biology.Synanthrin, also known as inulin are a kind of wide
The general stem tuber for being present in jerusalem artichoke, Ji root in polysaccharide.Synanthrin molecule is by 30 or so beta-D-fructofuranoses and 1~2 pyrans
Glucose residue is polymerized, and is by D-Fructose through linear straight chain polysaccharide made of β (1-2) glycosidic bond links, and end often carries
One glucose residue.Synanthrin has control blood glucose, blood fat, adjusts the physiological activity such as intestinal microflora, in biology, doctor
The fields such as, health care have extensive use.
It is common using citric acid as carbon source, the big multi-emitting blue-fluorescence of carbon quantum dot of hydrothermal synthesis, such as Shoujun Zhu
Deng (Shoujun Zhu, Qingnan Meng, Lei Wang, Junhu Zhang, Yubin Song, Han Jin, Kai
Zhang, Hongchen Sun, Haiyu Wang, Bai Yang., Angew.Chem.2013,125,4045-4049) report
Method, and the launch wavelength of such carbon quantum dot is typically to change with the variation of excitation wavelength, that is, has excitation wavelength
Dependence.
Dextrase is a kind of multiple-microorganism in plant, soil, animal alimentary canal, can hydrolyze β (1-2)
The hydrolase of glycosidic bond, scientific name are β (1-2)-D levanases.Dextrase can cut list one by one from the non-reducing end of levulan
Sugared (5 prime excision enzyme activity), can also cut off some β (1-2) glycosidic bond (endonuclease activity) at random in levulan intramolecule.People
Body itself can not synthesize dextrase, need to generate chrysanthemum by the various microorganism species such as Escherichia coli, Bifidobacterium in enteron aisle
Carbohydrase, resulting dextrase also promote intestinal microflora more preferable while contributing to absorption of human body to utilize synanthrin
The a variety of biochemical functions of performance, therefore, dextrase level of activity is significant to human body intestinal canal microorganism species.However it passes through
Investigation finds that existing dextrase activity detection technique means are deficient, lack quickly and accurately dextrase activity detection method.Therefore
It explores and realizes rapidly and efficiently detection to dextrase activity, be of great significance and good prospect in life science.
Invention content
In order to solve the deficiencies in the prior art, an object of the present invention is to provide a kind of for the detection of dextrase activity
Carbon quantum dot dextrase activity can detect, fill up dextrase activity determination technological gap in aqueous solution, and with it is easy,
Quickly, the features such as efficient, is of great significance and good prospect in life science.
To achieve the goals above, the technical scheme is that：
A kind of carbon quantum dot for the detection of dextrase activity is prepared using synanthrin and citric acid as carbon source, obtained
Carbon quantum dot can emit the green fluorescence that maximum wavelength is 520nm.
The carbon quantum dot of the present invention can not only detect the dextrase activity in aqueous solution, and emit the wavelength of fluorescence not
With excitation wavelength and change.
The second object of the present invention is to provide a kind of preparation method of the carbon quantum dot for the detection of dextrase activity, with chrysanthemum
Sugar and citric acid are carbon source, and carrying out reaction using hydro-thermal method can be obtained carbon quantum dot；The temperature of hydro-thermal method is 150~180 DEG C,
Reaction time is 3~6h.
The third object of the present invention is to provide the carbon quantum dot that a kind of above-mentioned carbon quantum dot or above-mentioned preparation method obtain and exists
It is applied in detection dextrase activity.
The fourth object of the present invention is to provide a kind of detection method in detection dextrase activity, by above-mentioned carbon quantum dot or
The carbon quantum dot that above-mentioned preparation method obtains is as fluorescence probe.
Preferably a kind of detection method in detection dextrase activity of the present invention, by above-mentioned carbon quantum dot or above-mentioned preparation method
The carbon quantum dot of acquisition is dissolved in the water as fluorescence probe and prepares several pieces carbon quantum dot aqueous solution, then respectively to several carbon amounts
The dextrase of different specific activity is added in son point aqueous solution, prepares the dextrase standard solution of different activity, detects synanthrin
The fluorescence intensity of enzyme standard solution corresponds to detected fluorescence intensity fitting according to different activity and draws standard curve, according to
Standard curve and detection to actual sample, to realize the activity detection of dextrase.
Beneficial effects of the present invention are：
1) present invention explores new carbon quantum dot and prepares material, and the carbon quantum dot fluorescence property of preparation is good, transmitted wave
Length does not change with the change of excitation wavelength, and convenient and simple for operation easy, and raw material are nontoxic, are easy to get, and can be widely applied to more
The fields such as kind Molecular Detection, biological medicine, optical device.
2) a kind of method that synanthrin and citric acid disclosed by the invention prepare carbon quantum dot and detected for dextrase activity,
Be successfully applied in actual sample dextrase activity to detect, fill up dextrase activity determination technological gap, have quickly, efficiently,
The features such as environmentally protective, has applications well foreground.
Description of the drawings
The accompanying drawings which form a part of this application are used for providing further understanding of the present application, and the application's shows
Meaning property embodiment and its explanation do not constitute the improper restriction to the application for explaining the application.
Fig. 1 is the design drawing that carbon quantum dot of the present invention is prepared and detected；
Fig. 2 is the emission spectrum of carbon quantum dot prepared by embodiment 1；
The fluorescence emission spectrum after different activity dextrases is added in the positions Fig. 3.
Specific implementation mode
It is noted that described further below be all exemplary, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific implementation mode, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or combination thereof.
Hydro-thermal method described herein refers in the pressure vessel of sealing, using water as solvent, in the condition of high temperature and pressure
The chemical reaction of lower progress.It is 100~1000 DEG C that high temperature, which refers to temperature, and high pressure finger pressure is 1MPa~1GPa.
As background technology is introduced, the deficiency of dextrase activity detection technique means scarcity exists in the prior art,
In order to solve technical problem as above, present applicant proposes a kind of carbon quantum dots and preparation method for the detection of dextrase activity
And application.
A kind of exemplary embodiment of the application provides a kind of carbon quantum dot for the detection of dextrase activity, with chrysanthemum
Sugar and citric acid are prepared by carbon source, and carbon quantum dot obtained can emit the green fluorescence that maximum wavelength is 520nm.
The carbon quantum dot of the present invention can not only detect the dextrase activity in aqueous solution, and emit the wavelength of fluorescence not
With excitation wavelength and change.
The another embodiment of the application provides a kind of preparation side of the carbon quantum dot for the detection of dextrase activity
Method carries out reaction using hydro-thermal method and can be obtained carbon quantum dot using synanthrin and citric acid as carbon source；The temperature of hydro-thermal method is 150
~180 DEG C, the reaction time is 3~6h.
Preferably, the mass ratio of synanthrin and citric acid is 0.02~0.1:0.2~1.
Preferably, the input ratio of synanthrin, citric acid and water is 0.02~0.1:0.2~1:10~50, g:g:mL.
Preferably, the volume of water and the volumetric ratio of pressure vessel are 2 in hydro-thermal method:4~5.
In order to obtain pure carbon quantum dot, the application is preferred, is first centrifuged, is filtered after being reacted using hydro-thermal method
Insoluble bulky grain is removed, then filtrate is subjected to dialysis removal unreacted reactant, is extracted after removing solvent, is then centrifuged for being precipitated
Object, and will be after drying precipitate.
It is further preferred that the centrifugal condition for removing insoluble bulky grain is, and 6000~12000rpm of centrifugal rotational speed, centrifugation
2~10min of time.
It is further preferred that removing the filtering of insoluble bulky grain using 0.22 μm of nylon leaching film.
It is further preferred that dialysis uses the molecular cut off of dialysis membrane for 1800Da.Dialysis time is 12~48h.
It is further preferred that the mode that removal solvent uses is rotary evaporation.The temperature of rotary evaporation is 50~80 DEG C.
It is further preferred that the condition that centrifugation obtains sediment is 8000~12000rpm of centrifugal rotational speed, centrifugation time 5
~15min.
It is further preferred that dry mode is freeze-drying.The temperature and time of freeze-drying is respectively -70~-50
DEG C and 12~48h.
Embodiment there is provided the carbon amounts that a kind of above-mentioned carbon quantum dot or above-mentioned preparation method obtain for the third of the application
Son point is applied in detecting dextrase activity.
Embodiment there is provided a kind of detection methods in detection dextrase activity for the 4th kind of the application, by above-mentioned carbon
The carbon quantum dot that quantum dot or above-mentioned preparation method obtain is as fluorescence probe.
Preferably a kind of detection method in detection dextrase activity of the present invention, by above-mentioned carbon quantum dot or above-mentioned preparation method
The carbon quantum dot of acquisition is dissolved in the water as fluorescence probe and prepares several pieces carbon quantum dot aqueous solution, then respectively to several carbon amounts
The dextrase of different specific activity is added in son point aqueous solution, prepares the dextrase standard solution of different activity, detects synanthrin
The fluorescence intensity of enzyme standard solution corresponds to detected fluorescence intensity fitting according to different activity and draws standard curve, according to
Standard curve and detection to actual sample, to realize the activity detection of dextrase.
It is further preferred that a concentration of 0.50~5.00mg/mL of carbon quantum dot aqueous solution.
It is further preferred that the specific activity of the dextrase of different specific activity be respectively 0U/mL, 4U/mL, 20U/mL,
40U/mL。
It is further preferred that dextrase standard solution constant temperature is detected fluorescence intensity again after for a period of time.The temperature of constant temperature
Degree and time are respectively 40~60 DEG C and 1~10h.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
Embodiment 1
The preparation process and detection process of carbon quantum dot are as shown in Figure 1.
It weighs synanthrin 0.02g and citric acid 0.20g is dissolved in 10mL deionized waters, 4min is to uniform for stirring, is placed in
Hydro-thermal reaction in 20mL autoclaves, setting reaction temperature are 150 DEG C, reaction time 3h, and reactant is centrifuged in 6000rpm
5min, 0.22 μm of nylon leaching film filtering remove insoluble bulky grain, not in molecular cut off 1800Da dialysis membranes dialysis 20h removals
Reactant, 60 DEG C of rotary evaporations remove solvent, acetone extract are added, and lower sediment thing is obtained with 8000rpm centrifugations 5min, be placed in-
50 DEG C of freeze-drying 20h, obtain carbon quantum dot.
Characterized that the results are shown in Figure 2 to the carbon quantum dot of preparation, it can be seen that the carbon quantum dot fluorescence of preparation
Can be good, launch wavelength does not change with the change of excitation wavelength.
Carbon quantum dot obtained is dissolved in deionized water and does fluorescence probe in a concentration of 0.50mg/mL, is separately added into chrysanthemum
Carbohydrase 0U/mL, 4U/mL, 20U/mL, 40U/mL, 3min is to uniform for stirring, is placed in 40 DEG C of waters bath with thermostatic control after reacting 1h,
6000rpm centrifugation 5min after take supernatant fluorescence intensity, as shown in figure 3, fitting carbon quantum dot fluorescence intensity decreasing value with
Dextrase activity linear relationship draws standard curve, carries out actual sample detection, realizes the activity detection of dextrase.
Embodiment 2
It weighs synanthrin 0.05g and citric acid 0.50g is dissolved in 20mL deionized waters, 7min is to uniform for stirring, is placed in
Hydro-thermal reaction in 50mL autoclaves, setting reaction temperature are 160 DEG C, reaction time 5h, and reactant is centrifuged in 8000rpm
8min, 0.22 μm of nylon leaching film filtering remove insoluble bulky grain, not in molecular cut off 1800Da dialysis membranes dialysis 30h removals
Reactant, 70 DEG C of rotary evaporations remove solvents, and acetone extract is added, with 10000rpm centrifuge 10min lower sediment thing,
- 60 DEG C of freeze-drying 30h are placed in, carbon quantum dot is obtained.
Carbon quantum dot obtained is dissolved in deionized water and does fluorescence probe in a concentration of 1.00mg/mL, is separately added into chrysanthemum
Carbohydrase 0U/mL, 4U/mL, 20U/mL, 40U/mL, 5min is to uniform for stirring, is placed in 50 DEG C of waters bath with thermostatic control after reacting 2h,
Supernatant fluorescence intensity, fitting carbon quantum dot fluorescence intensity decreasing value and dextrase activity are taken after 8000rpm centrifugations 5min
Linear relationship draws standard curve, carries out actual sample detection, realizes the activity detection of dextrase.
Embodiment 3
It weighs synanthrin 0.10g and citric acid 0.80g is dissolved in 50mL deionized waters, 10min is to uniform for stirring, is placed in
Hydro-thermal reaction in 100mL autoclaves, setting reaction temperature are 180 DEG C, reaction time 6h, and reactant is centrifuged in 10000rpm
10min, 0.22 μm of nylon leaching film filtering remove insoluble bulky grain, in the dialysis 40h removals of molecular cut off 1800Da dialysis membranes
Unreacted reactant, 80 DEG C of rotary evaporations remove solvents, and acetone extract is added, with 10000rpm centrifuge 10min lower sediment
Object is placed in -65 DEG C of freeze-drying 48h, obtains carbon quantum dot.
Carbon quantum dot obtained is dissolved in deionized water and does fluorescence probe in a concentration of 3.00mg/mL, is separately added into chrysanthemum
Carbohydrase 0U/mL, 4U/mL, 20U/mL, 40U/mL, 10min is to uniform for stirring, is placed in 55 DEG C of waters bath with thermostatic control after reacting 5h,
Supernatant fluorescence intensity, fitting carbon quantum dot fluorescence intensity decreasing value and dextrase activity are taken after 10000rpm centrifugations 5min
Linear relationship draws standard curve, carries out actual sample detection, realizes the activity detection of dextrase.
Embodiment 4
It weighs synanthrin 0.10g and citric acid 1.00g is dissolved in 50mL deionized waters, 10min is to uniform for stirring, is placed in
Hydro-thermal reaction in 100mL autoclaves, setting reaction temperature are 180 DEG C, reaction time 6h, and reactant is centrifuged in 12000rpm
10min, 0.22 μm of nylon leaching film filtering remove insoluble bulky grain, in the dialysis 48h removals of molecular cut off 1800Da dialysis membranes
Unreacted reactant, 80 DEG C of rotary evaporations remove solvents, and acetone extract is added, with 12000rpm centrifuge 10min lower sediment
Object is placed in -65 DEG C of freeze-drying 48h, obtains carbon quantum dot.
Carbon quantum dot obtained is dissolved in deionized water and does fluorescence probe in a concentration of 5.00mg/mL, is separately added into chrysanthemum
Carbohydrase 0U/mL, 4U/mL, 20U/mL, 40U/mL, 10min is to uniform for stirring, is placed in 50 DEG C of waters bath with thermostatic control after reacting 10h,
Supernatant fluorescence intensity, fitting carbon quantum dot fluorescence intensity decreasing value and dextrase activity are taken after 12000rpm centrifugations 5min
Linear relationship draws standard curve, carries out actual sample detection, realizes the activity detection of dextrase.
The foregoing is merely the preferred embodiments of the application, are not intended to limit this application, for the skill of this field
For art personnel, the application can have various modifications and variations.Within the spirit and principles of this application, any made by repair
Change, equivalent replacement, improvement etc., should be included within the protection domain of the application.
Claims (10)
1. a kind of carbon quantum dot for the detection of dextrase activity, characterized in that it is prepared as carbon source using synanthrin and citric acid,
Carbon quantum dot obtained can emit the green fluorescence that maximum wavelength is 520nm.
2. a kind of preparation method of carbon quantum dot for the detection of dextrase activity, characterized in that using synanthrin and citric acid as carbon
Source carries out reaction using hydro-thermal method and can be obtained carbon quantum dot；The temperature of hydro-thermal method be 150~180 DEG C, the reaction time be 3~
6h。
3. the preparation method as shown in claim 2, characterized in that the mass ratio of synanthrin and citric acid is 0.02~0.1:0.2
~1.
4. the preparation method as shown in claim 2, characterized in that the input ratio of synanthrin, citric acid and water is 0.02~0.1:
0.2~1:10~50, g:g:mL.
5. the preparation method as shown in claim 2, characterized in that the volume of water and the volumetric ratio of pressure vessel are in hydro-thermal method
2:4~5.
6. the preparation method as shown in claim 2, characterized in that first centrifuged, filtered after being reacted using hydro-thermal method
Insoluble bulky grain is removed, then filtrate is subjected to dialysis removal unreacted reactant, is extracted after removing solvent, is then centrifuged for being precipitated
Object, and will be after drying precipitate；
Preferably, the centrifugal condition for removing insoluble bulky grain is 6000~12000rpm of centrifugal rotational speed, centrifugation time 2~
10min；
Preferably, dialysis uses the molecular cut off of dialysis membrane for 1800Da；
Preferably, the condition that centrifugation obtains sediment is 8000~12000rpm of centrifugal rotational speed, 5~15min of centrifugation time；
Preferably, dry mode is freeze-drying, the temperature and time of freeze-drying is respectively -70~-50 DEG C and 12~
48h。
7. the carbon quantum that a kind of carbon quantum dot described in claim 1 or any preparation method of claim 2~6 obtain
Point is applied in detecting dextrase activity.
8. a kind of detection method in detection dextrase activity, characterized in that by carbon quantum dot described in claim 1 or right
It is required that the carbon quantum dot that 2~6 any preparation methods obtain is as fluorescence probe.
9. detection method as claimed in claim 8, characterized in that by carbon quantum dot described in claim 1 or claim 2
The carbon quantum dot that~6 any preparation methods obtain is dissolved in the water as fluorescence probe prepares several pieces carbon quantum dot water
Solution, then the dextrase of different specific activity is added to several carbon quantum dot aqueous solutions respectively, prepare the chrysanthemum of different activity
Carbohydrase standard solution detects the fluorescence intensity of dextrase standard solution, detected fluorescence intensity is corresponded to according to different activity
Standard curve is drawn in fitting, according to standard curve and to the detection of actual sample, to realize the activity detection of dextrase.
10. detection method as claimed in claim 9, characterized in that a concentration of 0.50~5.00mg/ of carbon quantum dot aqueous solution
mL；
Or, the specific activity of the dextrase of different specific activity is respectively 0U/mL, 4U/mL, 20U/mL, 40U/mL；
Or, dextrase standard solution constant temperature is detected fluorescence intensity again after for a period of time, the temperature and time of constant temperature is respectively
40~60 DEG C and 1~10h.
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