CN109444103B - Preparation method of PEI functionalized green fluorescent carbon dots and thrombin detection method based on carbon dots - Google Patents
Preparation method of PEI functionalized green fluorescent carbon dots and thrombin detection method based on carbon dots Download PDFInfo
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Abstract
The invention discloses a preparation method of PEI functionalized green fluorescent carbon dots and a thrombin detection method based on the carbon dots, wherein malic acid and Polyethyleneimine (PEI) are adopted as precursors, and the PEI functionalized green fluorescent carbon dots are prepared in one step by a hydrothermal method, so that the method is simple and easy to implement, the raw materials are cheap and easy to obtain, the cost is low, the yield is high, the method is suitable for batch production, and the prepared carbon dots have emission wavelengths; the PEI functionalized carbon dots with high cation density can improve the luminous efficiency and the dispersibility of the carbon dots, and simultaneously can ensure that the surfaces of the carbon dots have rich positive charges and the quantum yield is high; the thrombin detection method based on the green fluorescent carbon dots has the advantages that the carbon dots and the aptamers are not modified, and a label-free method is adopted, so that the synthesis steps are simplified to a great extent, and the number of the synthetic steps is reduced; has the characteristics of low operation cost, simple preparation process, mild reaction condition and easy popularization.
Description
Technical Field
The invention belongs to the technical field of fluorescent carbon nano materials and biosensing, and relates to a preparation method of PEI functionalized green fluorescent carbon dots and a thrombin detection method based on the carbon dots.
Background
The carbon dots are a novel zero-dimensional carbon nano material. Besides excellent fluorescence property, the carbon dots also have the advantages of low toxicity, good biocompatibility, simple and mild preparation steps, easy surface modification, abundant and cheap raw materials and the like. Therefore, the carbon dots can be applied to the biomedical field as substitutes for semiconductor quantum dots and organic dyes.
The existing carbon dot synthesis methods mainly comprise a laser ablation method, an electrochemical method, a pyrolysis method, an acid oxidation method, a microwave method, an ultrasonic method and a hydrothermal method. The carbon dot properties obtained by different preparation methods are different. However, most of the carbon dots prepared by the methods are limited to short-wavelength luminescence, most of the carbon dots emit blue fluorescence, cells or tissues of organisms have strong self-blue fluorescence, and when the carbon dots are used for cell imaging or biological component measurement, the differentiation of target signals and background signals is not facilitated, and the background interference is large.
Although reports on long-wavelength fluorescent carbon dots have been made in recent two years, the prospective work still has many problems to be solved, which mainly show the defects of low emission efficiency and poor water solubility of the fluorescence of the carbon dots in the long-wavelength region (more than 500nm), thereby limiting the further development and application of the carbon dots in the fields of biomedicine and the like. Therefore, the preparation of carbon dots with long wavelength and high fluorescence quantum yield is a constantly pursued goal.
Thrombin is a multifunctional serine protease and plays an important role in molecular biology, such as blood coagulation, angiogenesis, diagnosis of tumor growth and metastasis, and the like. In addition, excessive blood coagulation can cause diseases such as thromboembolism in vivo and even death of the organism. Therefore, establishing a simple and rapid method for detecting thrombin content is of great significance to early diagnosis, disease course development, prognosis, monitoring and evaluation of curative effect and the like of clinical diseases.
The aptamer is an oligonucleotide sequence, can be combined with a target in a high specificity and high affinity manner through a specific three-dimensional structure, has a wide target molecule range, and can specifically recognize small molecules and proteins and even can be combined with the whole cell. This indicates that the aptamer as a recognition element can significantly broaden the application range of the relevant sensor. In recent years, aptamer-based fluorescence biosensors have become a potential alternative to traditional detection methods. A series of fluorescent aptamer sensors for thrombin detection have been reported. However, these methods often require chemical modification of aptamers or labeling of aptamers with advanced nanomaterials, fluorescein, etc. as fluorophores, which is time-consuming and complicated, increases the cost, and reduces the affinity of aptamers to the target. Therefore, it is important to develop a non-labeled fluorescence aptamer sensor for thrombin detection.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a preparation method of PEI functionalized green fluorescent carbon dots with strong green fluorescence and a thrombin detection method based on the carbon dots, wherein the prepared carbon dots have long wavelength and high quantum yield, and a novel thrombin detection method with high selectivity by taking positive charge carbon dots as fluorescent probes and aptamers as recognition probes is provided.
In order to achieve the purpose, the invention adopts the technical scheme that:
a preparation method of PEI functionalized green fluorescent carbon dots comprises the following steps:
(1) dissolving malic acid and PEI in ultrapure water according to the mass ratio of 1: 5-4: 1, uniformly stirring, placing in a reaction kettle, and carrying out hydrothermal reaction at 120-220 ℃ for 3-12 h to obtain a brown yellow solution;
(2) carrying out suction filtration on the brown yellow solution obtained in the step (1) to remove large-particle impurities, and carrying out vacuum drying on the filtrate to obtain solid powder;
(3) and (3) adding absolute ethyl alcohol into the solid powder obtained in the step (2), performing ultrasonic treatment, centrifuging, removing a supernatant, and performing freeze drying on a precipitate to obtain a purified carbon dot.
Further, the centrifugation rotating speed in the step (3) is 13000rpm-16000rpm, and the centrifugation time is 10-30 min.
Further, the aperture of the suction filtration membrane in the step (2) is 0.22 μm.
Further, the vacuum drying temperature in the step (2) is 50 ℃.
A thrombin detection method based on green fluorescent carbon dots comprises the following steps:
(1) and fluorescence quenching: adding the thrombin aptamer into the carbon dot dispersion liquid to prepare a P-CDs-aptamer sensor for detecting thrombin; aptamers can quench the fluorescence of positively charged carbon dots (P-CDs);
(2) and (3) detecting thrombin: after thrombin is added and the aptamer is combined with thrombin, the fluorescence of the positive charge carbon dots (P-CDs) is recovered, and the detection of the thrombin is realized according to the change of the recovery intensity of the fluorescence.
Further, in the step (2), the carbon dots with positive charge (P-CDs), thrombin aptamer, NaCl, MgCl and the like are added in sequence in the EP tube2And (3) reacting with a phosphate buffer solution for 30min, then adding a thrombin solution, incubating for a period of time, and carrying out fluorescence intensity determination on the incubated solution.
Further, positively charged carbon sites (P-CDs) were mixed with thrombin aptamer in phosphate buffer with phosphate solution concentration of 10mM and NaCl concentration of 100 mM; mg (magnesium)2+The concentration is 4mM and the thrombin concentration is 1-200 nM.
Further, the incubation time was 40 min.
Compared with the prior art, the invention has the following advantages:
according to the preparation method of the PEI functionalized green fluorescent carbon dot, malic acid and Polyethyleneimine (PEI) are used as precursors, and the PEI functionalized green fluorescent carbon dot is prepared in one step by a hydrothermal method.
1. The synthesis method is simple: the green fluorescent carbon dot preparation method can obtain the green fluorescent carbon dot by simple one-step hydrothermal synthesis of two molecules, is simple and easy to implement, low in cost and high in yield, is suitable for batch production, and has the advantages of cheap and easily-obtained raw materials, and the prepared carbon dot has the emission wavelength of 502nm, wherein the maximum emission wavelength is shown in figure 3. Most of the carbon dots prepared by the methods reported in the literature at present emit blue fluorescence under the excitation of ultraviolet light, and the main emission peak is 410-440 nm.
2. Good water solubility and high fluorescence quantum yield: the PEI functionalized carbon dots with high cationic density are prepared by malic acid and Polyethyleneimine (PEI), so that the luminous efficiency and the dispersibility of the carbon dots are improved, the surfaces of the carbon dots are provided with abundant positive charges, and the quantum yield is high.
The thrombin detection method based on the green fluorescent carbon dots is characterized in that a positive charge carbon dot (P-CDs) solution has good fluorescence emission property, when a thrombin aptamer is added into the P-CDs solution, the aptamer with negative charge is adsorbed to the surface of the P-CDs due to the electrostatic interaction between the aptamer and the positive charge carbon dot to quench the fluorescence of the carbon dot, thrombin is added into an aptamer/P-CDs compound system, and the thrombin is combined with the aptamer to be away from the surface of the P-CDs, so that the fluorescence of the P-CDs is recovered, the degree of the fluorescence recovery of the P-CDs is gradually enhanced along with the increase of the concentration of the thrombin, and the fluorescence intensity of the P-CDs is in a linear relation with the concentration of the thrombin, so that the thrombin detection method based on the green fluorescent carbon dots can be used for sensitive and selective detection of the thrombin.
1. The method does not need to be marked, and reduces the detection cost: in the invention, both the carbon points and the aptamers are not modified, and a label-free method is adopted, so that the synthesis steps are simplified to a great extent, and simultaneously, the number of the carbon points and the aptamers is reduced; has the characteristics of low operation cost, simple preparation process, mild reaction condition and easy popularization.
2. The sensitivity and the selectivity are high: the invention adopts an off-on fluorescence detection mode, utilizes strong specific binding between thrombin and an aptamer, and can obviously improve the sensitivity and selectivity of detection.
3. The accuracy is high: during detection, the carbon dots and the aptamers do not need any modification, and the external interference is reduced to a great extent when the carbon dots and the aptamers are applied to quantitative detection of thrombin.
4. The practicability is strong: the detection method is a common detection method, and can realize the detection of other substrate molecules only by changing the sequence of the aptamer.
Drawings
FIG. 1 is a TEM photograph of green P-CDs prepared in example 1 of the present invention
FIG. 2 is a particle size distribution chart of green P-CDs prepared in example 1 of the present invention
FIG. 3 is a graph showing UV-VIS absorption spectrum and fluorescence emission spectrum of green P-CDs prepared in example 1 of the present invention
FIG. 4 is a fluorescence emission spectrum of green P-CDs prepared in example 1 of the present invention at different excitation wavelengths (310-450nm)
FIG. 5 is an infrared spectrum of green P-CDs prepared in example 1 of the present invention
FIG. 6 is a graph showing fluorescence spectra of aptamer/P-CDs complex and thrombin at different concentrations
FIG. 7 is a graph of selectivity for thrombin detection.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the invention thereto.
Example 1 preparation method of PEI functionalized green fluorescent carbon dots:
(1) dissolving 0.5g of PEI and 0.5g of malic acid in 13mL of ultrapure water, uniformly stirring, placing in a reaction kettle, and carrying out hydrothermal reaction for 10 hours at 180 ℃ to obtain a brown yellow solution;
(2) filtering the brown yellow solution obtained in the step (1) through a filter membrane with the aperture of 0.22 mu m, removing large-particle impurities, and drying the filtrate in vacuum at 50 ℃ to obtain solid powder;
(3) and (3) adding absolute ethyl alcohol into the solid powder obtained in the step (2), centrifuging for 15min at the rotating speed of 16000r/min by using a centrifuge after ultrasonic treatment, removing supernatant, and dispersing the precipitate in ultrapure water again after freeze drying to obtain the P-CDs dispersion.
FIG. 1 is a transmission electron microscope photograph of the P-CDs obtained in example 1, from which it can be seen that the P-CDs are spherical or spheroidal and have good dispersibility without agglomeration.
FIG. 2 is a statistical graph of the particle size distribution of P-CDs obtained in example 1, and it can be seen that the particle size distribution of the obtained P-CDs is between 1.2 and 5.1nm and the average particle size is 2.6 nm.
FIG. 3 is a UV-VIS absorption spectrum and a fluorescence emission spectrum of green P-CDs prepared in example 1, showing that the UV-VIS absorption spectrum of the P-CDs obtained in example 1 has a distinct absorption peak at 331 nm. The P-CDs emit green fluorescence at 502nm under the excitation wavelength of 430nm, and the fluorescence quantum yield of the P-CDs is measured to be 40.9% by taking quinine sulfate as a reference solution. The inset is a photograph of the P-CDs dispersion under visible light and 365nm ultraviolet light respectively, the P-CDs dispersion is clear and transparent yellow under the visible light, and emits bright green fluorescence under the irradiation of an ultraviolet lamp.
FIG. 4 is a fluorescence emission spectrum of green P-CDs at different excitation wavelengths. It can be seen that the excitation wavelength interval is 20nm, and in the range of the excitation light of 310-450nm, the emission wavelength is red-shifted with the red shift of the excitation light and is accompanied by the increase and then decrease of the fluorescence intensity, which may be related to the energy trap emission and electron conjugated structure of the P-CDs.
FIG. 5 is an infrared spectrum of P-CDs, malic acid and PEI obtained in example 1, from which it can be seen that in the P-CDs spectrum, PEI is involved and is located at 1453cm-1CH of (A)2In-plane bending vibration and vibration at 770cm-1The out-of-plane vibration of N-H disappears; corresponding to the carboxyl character 1692cm of malic acid-1The peak also disappeared; and at 3200--1The occurrence of acyl radicals due to hydrogen bondingPeak of characteristic wave number increase of amine (C ═ O) at 1587cm-11634cm of swellin NH bending vibration-1Generates primary amine N-H bending vibration at 1358cm-1The secondary amine C-N stretching vibration peak is generated, 1171cm-1Primary amine C-N stretching vibration peaks appear at the position. The infrared spectrum result of the P-CDs shows that a condensation product of malic acid and PEI exists in the P-CDs product.
Example 2 preparation of P-CDs:
the preparation procedure is the same as example 1, and 6P-CDs with different fluorescence quantum yields are prepared by only changing the ratio of malic acid to PEI, and are shown in Table 1:
example 3 preparation of P-CDs:
the preparation steps are the same as example 1, only the hydrothermal reaction time and temperature are changed, and the reaction is carried out for 12 hours at the temperature of 120 ℃, so that the P-CDs with different fluorescence intensities are prepared.
Example 4 preparation of P-CDs:
the preparation steps are the same as example 1, only the hydrothermal reaction time and temperature are changed, and the reaction is carried out for 3 hours at the temperature of 220 ℃, so that the P-CDs with different fluorescence intensities are prepared.
Example 5 detection of thrombin using green fluorescent carbon spots as follows:
mu.L of the P-CDs dispersion prepared in example 1, 300. mu.L of phosphate buffer solution (10 mM in concentration, pH7.4, containing 100mM NaCl, 4mM MgCl) was added to the centrifuge tube in this order2) After mixing uniformly, adding 88 μ L of 10 μ M aptamer solution as fluorescence quenching agent, adding ultrapure water to constant volume of 600 μ L, shaking up, and standing for 30 min. After the fluorescence quenching of the P-CDs reaches the equilibrium, a series of thrombin (final concentration of 0, 5nM, 10nM, 20nM, 50nM, 100nM, 150nM, 200nM, 260nM) with different concentrations as a fluorescence recovery agent is added, shaken and shaken uniformly, incubated at 37 ℃ for 40min, and then fluorescence measurement is carried out with the excitation wavelength set to 430 nM.
As can be seen from FIG. 6, the values of P-CDs at thrombin concentrations of 0,5,10,20,50,100,150,200,260nMFluorescence intensity, the fluorescence intensity of P-CDs gradually increased with increasing thrombin concentration (inset is a linear plot of thrombin concentration versus fluorescence intensity). And the thrombin concentration is in a good linear relationship with the fluorescence enhancement degree (delta F) of the system in a range of 5-200 nM. The linear regression equation is 119.2+8.6C (nM) and the linear correlation coefficient is R2The assay for thrombin was 1.2nM 0.9963, which allows for sensitive detection of thrombin.
Example 6 stability and reproducibility
3 batches of P-CDs prepared in parallel were each measured to have a relative standard deviation of 3.78%. The fluorescence intensity of the P-CDs taken from the same batch is basically unchanged after 20 times of repeated measurement, and the fluorescence intensity of the prepared P-CDs is basically unchanged after the prepared P-CDs are stored for 3 months at room temperature. The results show that the fluorescence detection system based on the P-CDs is relatively stable, the measurement repeatability is good, and the accuracy of the detection result and the practicability of the detection method can be ensured.
Example 7 selectivity of P-CDs/aptamer complex nanoprobes for thrombin detection.
As can be seen from FIG. 7, only thrombin can significantly restore P-CDs fluorescence, while none of the other proteins such as Bovine Serum Albumin (BSA), hemoglobin (Hb) and lysozyme (Lys) can restore P-CDs fluorescence. The results show that the method has good selectivity for thrombin detection.
The aptamer sequences used in the above experiments were:
Thrombin aptamer：5′-NH2-(CH2)6-GGTTGGTGTGGTTGG-3′。
finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the embodiments of the invention without departing from the spirit and scope of the invention, which is to be covered by the claims.
Claims (6)
1. A preparation method of PEI functionalized green fluorescent carbon dots is characterized by comprising the following steps:
(1) dissolving malic acid and PEI in ultrapure water according to the mass ratio of 1: 5-4: 1, uniformly stirring, placing in a reaction kettle, and carrying out hydrothermal reaction at 120-220 ℃ for 3-12 h to obtain a brown yellow solution;
(2) carrying out suction filtration on the brown yellow solution obtained in the step (1) to remove large-particle impurities, and carrying out vacuum drying on the filtrate to obtain solid powder;
(3) adding absolute ethyl alcohol into the solid powder obtained in the step (2), performing ultrasonic treatment, centrifuging, removing supernate, and performing freeze drying on the precipitate to obtain purified carbon dots; the centrifugal speed is 13000rpm-16000rpm, and the centrifugal time is 10-30 min.
2. The method of claim 1, wherein: and (3) the aperture of the suction filtration membrane in the step (2) is 0.22 mu m.
3. The method of claim 1, wherein: the vacuum drying temperature in the step (2) is 50 ℃.
4. A thrombin detection method based on the green fluorescent carbon dot prepared in claim 1, which is characterized by comprising the following steps:
(1) and fluorescence quenching: adding the thrombin aptamer into the carbon dot dispersion liquid to prepare a P-CDs-aptamer sensor for detecting thrombin; aptamers can quench the fluorescence of positively charged carbon dots (P-CDs); mixing positive charge carbon dots (P-CDs) with a thrombin aptamer in a phosphate buffer solution, wherein the concentration of a phosphate solution in the phosphate buffer solution is 10mM, and the concentration of NaCl in the phosphate buffer solution is 100 mM; mg (magnesium)2+The concentration is 4mM, and the thrombin concentration is 1-200 nM;
(2) and (3) detecting thrombin: after thrombin is added and the aptamer is combined with thrombin, the fluorescence of the positive charge carbon dots (P-CDs) is recovered, and the detection of the thrombin is realized according to the change of the recovery intensity of the fluorescence.
5. According to the claimsThe thrombin detection method according to claim 4, wherein: in the step (2), positive charge carbon dots (P-CDs), thrombin aptamer, NaCl and MgCl are added into an EP tube in sequence2And (3) reacting with a phosphate buffer solution for 30min, then adding a thrombin solution, incubating for a period of time, and carrying out fluorescence intensity determination on the incubated solution.
6. The method for detecting thrombin according to claim 5, wherein: the incubation time was 40 min.
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