CN109444103A - A kind of PEI functionalization green fluorescent carbon dots preparation method and the blood coagulation enzyme assay method based on the carbon dots - Google Patents
A kind of PEI functionalization green fluorescent carbon dots preparation method and the blood coagulation enzyme assay method based on the carbon dots Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 28
- 230000023555 blood coagulation Effects 0.000 title claims abstract description 19
- 108010043121 Green Fluorescent Proteins Proteins 0.000 title claims abstract description 18
- 238000001952 enzyme assay Methods 0.000 title claims abstract description 14
- 238000007306 functionalization reaction Methods 0.000 title claims abstract description 13
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- 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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Abstract
The present invention discloses a kind of PEI functionalization green fluorescent carbon dots preparation method and the blood coagulation enzyme assay method based on the carbon dots, malic acid and polyethyleneimine (PEI) is used to prepare the green fluorescent carbon dots of PEI functionalization using one step of hydro-thermal method for presoma, method is simple and easy, raw material is cheap and easy to get, at low cost, output is high, suitable for mass production, the carbon dots launch wavelength of preparation is long；The present invention is the PEI functionalization carbon dots of high-cation density, and the luminous efficiency and dispersibility of carbon dots not only can be improved, while carbon dots surface can also be made high with positive charge abundant, quantum yield；Blood coagulation enzyme assay method based on green fluorescent carbon dots, carbon dots and aptamers are unmodified, using label-free method, significantly simplify synthesis step, reduce simultaneously；It is low with operating cost, preparation process is simple, the mild feature easy to spread of reaction condition.
Description
Technical field
The invention belongs to fluorescence carbon nanomaterials, biosensor technique field, are related to a kind of PEI functionalization green fluorescence carbon
Point preparation method and the blood coagulation enzyme assay method based on the carbon dots.
Background technique
Carbon dots are a kind of novel zero dimension carbon nanomaterials.In addition to excellent fluorescence property, carbon dots also have toxicity
Low, good biocompatibility, preparation step are simple mildly, are easy to the advantages that surface modification and abundant raw material are cheap.Therefore, carbon dots
It can be used as the substitute of semiconductor-quantum-point and organic dyestuff and be applied to field of biomedicine.
The synthetic method of carbon dots mainly has laser ablation method, electrochemical process, pyrolysismethod, acid oxidation, microwave method, surpasses at present
Sound method and hydro-thermal method.The carbon dots performance obtained using different preparation methods is also different.However, prepared by these methods
The carbon dots overwhelming majority is limited to short-wave long light-emitting, issues blue-fluorescence mostly, and the cell or tissue of organism have it is stronger from
Body blue-fluorescence is unfavorable for the area of target signal and background signal when these carbon dots are used for cell imaging or biological composition measurement
Point, background interference is big.
Although over the past two years about the report of long wavelength's fluorescent carbon point, these perspective work are still had
Many urgent problems to be solved, be mainly manifested in the fluorescence of carbon dots long wavelength region (being greater than 500nm) emission effciency it is low with
And the defect of poorly water-soluble, to limit carbon dots in the further development and application in the fields such as biomedicine.Therefore, long wave is prepared
The carbon dots of long high-fluorescence quantum yield are the targets that current people pursue always.
Fibrin ferment is a kind of Multi-functional wire serine protease, is examined in blood clotting, angiogenesis, growth and metastasis of tumours
Disconnected wait plays an important role in molecular biology.In addition, blood coagulation excessively also results in the generation of the diseases such as internal thromboembolism,
It even will cause body death.Therefore, simple, quickly detection thrombin amount method is established to examine the early stage of clinical disease
Disconnected, disease, prognosis and the monitoring of curative effect and assessment etc. all have very important significance.
Aptamer is a kind of oligonucleotide sequence, by its specific three-dimensional structure can with target high specific,
Combine to high-affinity, and target molecule range is wide, can not only specific recognition small molecule, protein, or even can also with it is whole
A cell combination.This shows that aptamer can significantly widen the application range of related sensor as recognition component.In recent years
Come, the biological sensor based on aptamers has become the potential selection instead of traditional detection method.It is a series of to be used for blood coagulation
The fluorescence aptamer sensor of enzyme detection has been reported.But these methods generally require to be chemically modified aptamers or utilize
Some advanced nano materials, fluorescein etc. come labeling nucleic acid aptamers, such modification and markers work as fluorophor
It is not only time-consuming, complicated, increase cost but also reduce aptamer to the affinity of object.Therefore, develop non-marked
Type fluorescence aptamer sensor detects particularly important for fibrin ferment.
Summary of the invention
To overcome above-mentioned the deficiencies in the prior art, the object of the present invention is to provide a kind of PEI for preparing intense green fluorescence
Functionalization green fluorescent carbon dots preparation method and blood coagulation enzyme assay method based on the carbon dots, the carbon dots long wavelength of preparation, quantum
Yield is high, and provides a kind of using positive charge carbon dots as fluorescence probe, the aptamers blood coagulation highly selective as identification probe
Enzyme new detecting method.
To achieve the above object, the technical solution adopted by the present invention is that:
A kind of PEI functionalization green fluorescent carbon dots preparation method, comprising the following steps:
(1), malic acid and PEI are dissolved in ultrapure water by 1:5~4:1 in mass ratio, are stirred evenly and are placed in reaction kettle,
3~12h of hydro-thermal reaction under the conditions of 120~220 DEG C, obtains brown yellow solution；
(2), the brown yellow solution for obtaining step (1) is by filtering, and removing large granular impurity, filtrate is vacuum dried,
Obtain solid powder；
(3), dehydrated alcohol is added in the solid powder obtained to step (2), after ultrasonic and is centrifuged, removes supernatant, sink
The carbon dots that freeze-drying of forming sediment is purified.
Further, centrifugal rotational speed is 13000rpm-16000rpm, centrifugation time 10-30min in the step (3).
Further, it is 0.22 μm that filter sizes are filtered in the step (2).
Further, vacuum drying temperature is 50 DEG C in the step (2).
A kind of blood coagulation enzyme assay method based on green fluorescent carbon dots, comprising the following steps:
(1), fluorescent quenching: thrombin aptamer is added in carbon dots dispersion liquid, that is, is prepared into the P- of detection fibrin ferment
CDs- aptamer sensor；Positive charge carbon dots (P-CDs) fluorescence is quenched in adaptation physical efficiency；
(2), the detection of fibrin ferment: being added fibrin ferment, after aptamers bind thrombin, positive charge carbon dots (P-CDs) it is glimmering
Light is restored, and according to the variation of fluorescence recovery strength, realizes the detection to fibrin ferment.
Further, in the step (2), by sequentially adding positive charge carbon dots (P-CDs) in EP pipe, fibrin ferment is adapted to
Body, NaCl, MgCl2, phosphate buffer solution add thrombin solution after reacting 30min, a period of time is incubated for, after incubation
Solution carry out fluorescent strength determining.
Further, positive charge carbon dots (P-CDs) are mixed with thrombin aptamer in phosphate buffer, phosphate buffer
Middle phosphate solution concentration is 10mM, NaCl concentration 100mM；Mg2+Concentration is 4mM, concentration of thrombin is 1~200nM.
Further, the incubation time is 40min.
Compared with prior art, the present invention has the advantage that
PEI functionalization green fluorescent carbon dots preparation method of the invention, uses malic acid and polyethyleneimine (PEI) for before
Drive the green fluorescent carbon dots that body prepares PEI functionalization using one step of hydro-thermal method.
1, synthetic method is simple: green fluorescent carbon dots preparation method of the invention passes through the simple step hydro-thermal of two molecules
Green fluorescent carbon dots can be obtained in synthesis, and method is simple and easy, raw material is cheap and easy to get, at low cost, output is high, are suitble to batch raw
It produces, the carbon dots launch wavelength of preparation is long, and maximum emission wavelength is located at 502nm, sees Fig. 3.And current most of sides reported in the literature
The carbon dots of method preparation launch blue-fluorescence under ultraviolet excitation, and main emission peak is in 410-440nm.
2. good water solubility, fluorescence quantum yield are high: high-cation is prepared using malic acid and polyethyleneimine (PEI)
The PEI functionalization carbon dots of density, not only can be improved the luminous efficiency and dispersibility of carbon dots, while can also make carbon dots surface band
There are positive charge abundant, quantum yield high.
A kind of blood coagulation enzyme assay method based on green fluorescent carbon dots, the blood coagulation based on green fluorescent carbon dots are provided simultaneously
Enzyme assay method, positive charge carbon dots (P-CDs) solution have good fluorescent emission property, thrombin aptamer are added to P-
When in CDs solution, negatively charged aptamers are adsorbed onto the surface P-CDs because of the electrostatic interaction between positive charge carbon dots and make
Carbon dots fluorescent quenching, is added fibrin ferment in aptamers/P-CDs complex systems, and fibrin ferment is combined to keep it remote with adaptation
From the surface P-CDs, the fluorescence of P-CDs is caused to restore, with the increase of concentration of thrombin, degree that the fluorescence of P-CDs restores by
Cumulative strong, the fluorescence intensity of P-CDs is in a linear relationship with concentration of thrombin, can be used for the sensitive of fibrin ferment and selectivity inspection
It surveys.
1. this method is without label, reduce testing cost: carbon dots and aptamers are unmodified in the present invention, use
Label-free method significantly simplifies synthesis step, reduces simultaneously；It is low with operating cost, preparation process is simple,
The mild feature easy to spread of reaction condition.
2. sensitivity, selectivity are high: present invention employs " off-on's " fluorescence detection modes, and fibrin ferment is utilized and fits
Strong specific binding between ligand, this system can significantly improve the sensitivity and selectivity of detection.
3. accuracy is high: carbon dots and aptamers are not necessarily to any modification when detection, are used in quantitative detection fibrin ferment very
Big degree reduces external interference.
4. practical: detection method is a kind of general character detection, need to only change the sequence of aptamer, so that it may
To realize the detection to other substrate molecules.
Detailed description of the invention
Fig. 1 is the transmission electron microscope photo of green P-CDs prepared by the embodiment of the present invention 1
Fig. 2 is the partial size statistical Butut of green P-CDs prepared by the embodiment of the present invention 1
Fig. 3 is the UV-visible absorption spectrum and fluorescence emission spectrum of green P-CDs prepared by the embodiment of the present invention 1
Figure
Fig. 4 be the embodiment of the present invention 1 prepare green P-CDs under different excitation wavelengths (310-450nm) fluorescence hair
Penetrate spectrogram
Fig. 5 is the infrared spectrogram of green P-CDs prepared by the embodiment of the present invention 1
Fig. 6 is aptamers/P-CDs compound and various concentration fibrin ferment fluorescence spectra
Fig. 7 is the selective figure detected to fibrin ferment.
Specific embodiment
Present invention is further described in detail combined with specific embodiments below, but not as a limitation of the invention.
Embodiment 1PEI functionalization green fluorescent carbon dots preparation method:
(1) 0.5g PEI and 0.5g malic acid are dissolved in 13mL ultrapure water, stir evenly and is placed in reaction kettle,
Hydro-thermal reaction 10h obtains brown yellow solution under the conditions of 180 DEG C；
(2) brown yellow solution for obtaining step (1) is filtered by the filter membrane that aperture is 0.22 μm, and it is miscellaneous to remove bulky grain
Matter, filtrate are dried in vacuo through 50 DEG C, obtain solid powder；
(3) dehydrated alcohol is added in the solid powder obtained to step (2), is turned after ultrasound with centrifuge with 16000r/min
Speed centrifugation 15min removes supernatant, is dispersed in ultrapure water again again after pellet frozen is dry, and resulting is P-CDs dispersion
Liquid.
Fig. 1 is the transmission electron microscope figure of 1 gained P-CDs of embodiment, and P-CDs is spherical or class ball as seen from the figure
Shape, and there is good dispersibility, do not reunite.
Fig. 2 is the particle diameter distribution statistical chart of 1 gained P-CDs of embodiment, it can be seen that the particle diameter distribution of gained P-CDs exists
Between 1.2-5.1nm, average grain diameter 2.6nm.
Fig. 3 is the UV-visible absorption spectrum and fluorescence emission spectrum of green P-CDs prepared by the embodiment of the present invention 1
Figure, it is seen that the uv-visible absorption spectra of 1 gained P-CDs of example, P-CDs have an apparent absorption peak in 331nm.?
P-CDs launches the green fluorescence of 502nm under 430nm excitation wavelength, uses quinine sulfate for reference solution, measures P-CDs's
Fluorescence quantum yield is 40.9%.Embedded figure is photo of the P-CDs dispersion liquid respectively under visible light and 365nm ultraviolet light,
P-CDs dispersion liquid is the yellow of clear under visible light, and bright green fluorescence is issued under ultraviolet light irradiation.
Fig. 4 is fluorescence emission spectrogram of compound of the green P-CDs under different excitation wavelengths.It can be seen that excitation wavelength spacing
It is launch wavelength red shift with the red shift of exciting light within the scope of 310-450nm in exciting light for 20nm, and strong along with fluorescence
Degree first increases to be reduced afterwards, and this phenomenon may emit related to electron conjugated structure to the energy trapping of P-CDs.
Fig. 5 is the infrared spectrogram of 1 gained P-CDs of example, malic acid and PEI, it can be seen that in P-CDs spectrum,
It is related with PEI and be located at 1453cm-1The CH at place2In-plane bending vibration and be located at 770cm-1The N-H out-of-plane vibration at place disappears；Accordingly
In malic acid carboxyl feature 1692cm-1Peak also disappears；And in 3200-3640cm-1Causing due to hydrogen bond action than one occurs in place
As the increased peak of amide (C=O) characteristic waves, while in 1587cm-1There is swollen amine NH bending vibration, 1634cm in place-1Place occurs
Primary amine N-H bending vibration, 1358cm-1There is secondary amine C-N stretching vibration peak, 1171cm-1There is primary amine C-N stretching vibration peak in place.
Above-mentioned p-CDs the results of FT-IR shows exist in P-CDs product by malic acid and PEI condensation product.
The preparation of embodiment 2P-CDs:
Preparation step only changes the P- of the different fluorescence quantum yields of 6 kinds of ratio preparation of malic acid and PEI with embodiment 1
CDs is shown in Table 1:
The preparation of embodiment 3P-CDs:
Preparation step only changes the hydro-thermal reaction time and temperature reacts 12h under the conditions of 120 DEG C, preparation is not with embodiment 1
With the P-CDs of fluorescence volume intensity.
The preparation of embodiment 4P-CDs:
Preparation step only changes the hydro-thermal reaction time and temperature reacts 3h under the conditions of 220 DEG C, preparation is not with embodiment 1
With the P-CDs of fluorescence volume intensity.
The blood coagulation enzyme assay method of the green fluorescent carbon dots of embodiment 5, specific as follows:
The 100 μ L of P-CDs dispersion liquid of the preparation of embodiment 1,300 μ L phosphate buffer solutions are sequentially added into centrifuge tube
(concentration 10mM, pH7.4, NaCl containing 100mM, 4mM MgCl2) after mixing, then 88 μ L, 10 μM of aptamers are added thereto
Solution is added ultrapure water and is settled to 600 μ L as fluorescence quenching, and concussion shakes up, and stands 30min.It is reached to P-CDs fluorescent quenching
To after balance, be added a series of various concentrations fibrin ferment (ultimate density 0,5nM, 10nM, 20nM, 50nM, 100nM,
150nM, 200nM, 260nM) it is used as fluorescence restorative, concussion shakes up, and after being incubated for 40min at 37 DEG C, setting excitation wavelength is
430nm carries out fluoremetry.
As seen from Figure 6, the fluorescence of P-CDs when concentration of thrombin is 0,5,10,20,50,100,150,200,260nM in figure
Intensity, with the increase of concentration of thrombin, the fluorescence intensity of P-CDs is gradually increased, and (interior illustration is that concentration of thrombin and fluorescence are strong
Linear relationship chart between degree).And concentration of thrombin is in good with system fluorescence enhancement degree (Δ F) within the scope of 5-200nM
Linear relationship.Equation of linear regression is Δ F=119.2+8.6C (nM), linearly dependent coefficient R2=0.9963, to blood coagulation
The detection line of enzyme is for 1.2nM, it can be achieved that the Sensitive Detection of fibrin ferment.
6 stability of embodiment and repeatability
The P-CDs for taking 3 criticize flat rows to prepare, measures, relative standard deviation 3.78% respectively.It takes with a batch P-CDs weight
Repetition measurement is 20 times fixed, and fluorescence intensity is basically unchanged, and is furthermore saved prepared P-CDs at room temperature 3 months, fluorescence
Also intensity does not change substantially.The above results show that the fluorescent detection system based on P-CDs is relatively stable, measurement repeatability
It is good, it can guarantee the accuracy of testing result and the practicability of detection method.
The selectivity that embodiment 7P-CDs/ adaptor complex nano-probe detects fibrin ferment.
As seen from Figure 7, only fibrin ferment just can be such that P-CDs fluorescence significantly restores, and other protein such as bovine serum albumin
(BSA), hemoglobin (Hb) and lysozyme (Lys) cannot be such that P-CDs fluorescence restores.The above result shows that the method for the present invention
There is good selectivity to fibrin ferment detection.
Aptamers sequence used in above-mentioned experiment are as follows:
Thrombin aptamer:5 '-NH2-(CH2)6-GGTTGGTGTGGTTGG-3′。
Finally it should be noted that: the above examples are only used to illustrate the technical scheme of the present invention rather than its limitations, to the greatest extent
Pipe is described the invention in detail referring to above-described embodiment, it should be understood by those ordinary skilled in the art that: still may be used
With modifications or equivalent substitutions are made to specific embodiments of the invention, and repaired without departing from any of spirit and scope of the invention
Change or equivalent replacement, should all cover in present claims range.
Claims (8)
1. a kind of PEI functionalization green fluorescent carbon dots preparation method, it is characterised in that the following steps are included:
(1), malic acid and PEI are dissolved in ultrapure water by 1:5~4:1 in mass ratio, are stirred evenly and are placed in reaction kettle,
3~12h of hydro-thermal reaction, obtains brown yellow solution under the conditions of 120~220 DEG C；
(2), the brown yellow solution for obtaining step (1) removes large granular impurity, filtrate is vacuum dried, obtains by filtering
Solid powder；
(3), dehydrated alcohol is added in the solid powder obtained to step (2), after ultrasonic and is centrifuged, it is cold to remove supernatant, precipitating
Freeze the carbon dots for being dried to obtain purifying.
2. preparation method according to claim 1, it is characterised in that: centrifugal rotational speed is 13000rpm- in the step (3)
16000rpm, centrifugation time 10-30min.
3. preparation method according to claim 1, it is characterised in that: filtering filter sizes in the step (2) is 0.22 μ
m。
4. preparation method according to claim 1, it is characterised in that: vacuum drying temperature is 50 DEG C in the step (2).
5. a kind of blood coagulation enzyme assay method of the green fluorescent carbon dots prepared based on claim 1, it is characterised in that including following
Step:
(1), fluorescent quenching: thrombin aptamer is added in carbon dots dispersion liquid, that is, is prepared into the P-CDs- of detection fibrin ferment
Aptamer sensor；Positive charge carbon dots (P-CDs) fluorescence is quenched in adaptation physical efficiency；
(2), the detection of fibrin ferment: being added fibrin ferment, and after aptamers bind thrombin, the fluorescence of positive charge carbon dots (P-CDs) is obtained
To restore, according to the variation of fluorescence recovery strength, the detection to fibrin ferment is realized.
6. blood coagulation enzyme assay method according to claim 5, it is characterised in that: in the step (2), by EP pipe
Sequentially add positive charge carbon dots (P-CDs), thrombin aptamer, NaCl, MgCl2, phosphate buffer solution, react 30min after,
Thrombin solution is added, a period of time is incubated for, the solution after incubation carries out fluorescent strength determining.
7. blood coagulation enzyme assay method according to claim 6, it is characterised in that: positive charge carbon dots (P-CDs) and fibrin ferment
Aptamers are mixed in phosphate buffer, in phosphate buffer phosphate solution concentration be 10mM, NaCl concentration 100mM；
Mg2+Concentration is 4mM, concentration of thrombin is 1~200nM.
8. blood coagulation enzyme assay method according to claim 6, it is characterised in that: the incubation time is 40min.
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