CN110257051B - Preparation method of DNA functionalized quantum dots based on click chemistry and application of DNA functionalized quantum dots in biomarker and detection - Google Patents

Preparation method of DNA functionalized quantum dots based on click chemistry and application of DNA functionalized quantum dots in biomarker and detection Download PDF

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CN110257051B
CN110257051B CN201910495182.7A CN201910495182A CN110257051B CN 110257051 B CN110257051 B CN 110257051B CN 201910495182 A CN201910495182 A CN 201910495182A CN 110257051 B CN110257051 B CN 110257051B
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何治柯
毛国斌
吉邢虎
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Wuhan University WHU
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Abstract

The invention discloses a preparation method of a DNA functionalized quantum dot based on click chemistry and application of the DNA functionalized quantum dot in biological labeling and detection. Relates to the field of nano materials and biomedical analytical chemistry. The preparation method comprises the step of adding DNA with one end modified by azide or diphenyl cyclooctyne and the other end modified by thiophosphate into the quantum dot synthesis process to serve as a co-stabilizer of the quantum dot, so as to obtain the azide or diphenyl cyclooctyne modified DNA functional quantum dot labeling reagent. The quantum dot labeling reagent can be used for efficiently labeling protease and cancer cells, and can be used for biological detection and imaging analysis. By combining the DNA hybridization principle, the labeling reagent also has important application value in the aspects of nano material self-assembly and the like. Meanwhile, compared with other fluorescence labeling reagents based on click chemistry, the preparation method of the labeling reagent is simple, does not need any chemical modification, is easy to purify and has excellent fluorescence performance.

Description

Preparation method of DNA functionalized quantum dots based on click chemistry and application of DNA functionalized quantum dots in biomarker and detection
Technical Field
The invention relates to the field of nano materials and biomedical analytical chemistry, in particular to an azide or diphenyl cyclooctyne modified DNA functionalized quantum dot and application thereof in the aspects of biomarker, detection and imaging.
Background
Quantum dots have excellent optical and chemical properties, such as adjustable emission spectrum, wide absorption range, large stokes shift, high quantum yield and good stability, and thus have been widely used in biosensing (i.l. Medintz et al, nat. mater.2005,4,435). In addition, quantum dots are also commonly used as labeling reagents for nucleic acids, proteins, viruses, and the like. Quantum dot labeling of proteins and the like has often employed a covalent coupling method, with carbodiimide methods being most commonly used. However, the carbodiimide method has problems such as low coupling efficiency, many by-products, easy hydrolysis, and poor label selectivity. Researchers have discovered a novel coupling modality, the bio-orthogonal reaction, also termed the click reaction (q.wang et al, j.am. chem. soc.2003,125, 3192). The first reported method is that copper ions catalyze cycloaddition reaction of azide and alkyne, and the method has good selectivity, high coupling efficiency, no hydrolysis by-product and no cross-coupling (J.E.Hein et al, chem.Soc.Rev.2010,39,1302). However, this method requires Cu+As a catalyst, excessive copper ions not only can damage cells and organisms, but also can quench fluorescence of quantum dots, so that the method has a certain limitation on labeling of fluorescent nano materials (a. bernardin et al, Bioconjugate chem.2010,21, 583). The tension-driven azide-alkyne cycloaddition reaction (SPAAC) has the advantages of no need of a catalyst, mild reaction conditions and the like (N.J.Agard et al, J.Am.chem. Soc.2004,126,15046) besides the advantage of catalyzing the azide-alkyne cycloaddition reaction by copper ions, and the method is widely applied in recent years.
Schieber et al labeled quantum dots on transferrin by SPAAC and achieved imaging analysis of tumor cells by the interaction of transferrin with transferrin receptors on the surface of tumor cells, with high labeling efficiency and without affecting protein activity (c.schieber et al, angelw.chem.int.edit.2012, 51, 10523). Hao et al use SPAAC methods to label quantum dots on envelope proteins of influenza viruses, which are mild and efficient (j.haoet al, anal.chem.2012,84,8364). The method needs to couple the diphenyl cyclooctyne compound on the surface of the quantum dot. The diphenyl cyclooctyne compound modification of the quantum dots is mainly realized by a carbodiimide method, the method can reduce the fluorescence efficiency of the quantum dots, and the stability can be influenced to a certain extent. Therefore, it is necessary to establish a simple and effective method for constructing a quantum dot labeling reagent based on a click reaction.
A class of DNA modified by phosphorothioate is designed by the Canadian Kelley topic group, and is introduced in the process of synthesizing quantum dots, and DNA functionalized quantum dots are synthesized by a one-pot method (N.Ma et al, nat. Nanotech. 2009,4, 121). The method does not need to modify the quantum dots, and can ensure that the luminous efficiency and stability of the quantum dots are not affected. Wherein, the DNA of the normal phosphate group still has targeting property and can react with target nucleic acid, protein, cells and the like. However, the quantum dots have only a single functionality for targeting.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a preparation method of an azide or diphenylcyclooctyne modified DNA functionalized quantum dot.
The invention further aims to provide application of the azide or diphenyl cyclooctyne modified DNA functionalized quantum dot in the aspects of biological labeling and detection. The labeling reagent is applied to labeling of glucose oxidase and HeLa cells, and shows certain universality. Compared with other covalent coupling methods, the probe for labeling the protease has the advantages of high connection efficiency, no by-product, high labeling speed, capability of enhancing the protease activity and the stability of quantum dots and the like; compared with other biological labeling methods, the probe for labeling HeLa cells has the advantages of no need of any chemical modification reagent, high labeling efficiency, high luminous efficiency and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing azide or diphenyl cyclooctyne modified DNA functionalized quantum dots comprises the following steps:
1) dissolving cadmium chloride, zinc chloride and N-acetyl-L-cysteine in deionized water to obtain a mixed solution A, and adjusting the pH value of the mixed solution A to 8.0-9.0 by using a sodium hydroxide solution; taking sodium tellurite and sodium borohydride in a molar ratio of 1:2, adding the sodium tellurite and sodium borohydride into a 10mL single-neck flask, placing the flask in an ice bath and under an anaerobic condition to react for 5 hours to obtain a mixed solution B, quickly transferring the mixed solution B into the mixed solution A, finally adding 20-200 mu M DNA modified by azide or diphenylcyclooctyne and thiophosphate, uniformly stirring the mixture, transferring the mixture into a hydrothermal reaction kettle, and reacting the mixture at 150-200 ℃ for 20-35 minutes to obtain the azide or diphenylcyclooctyne modified DNA functionalized CdTe, namely Zn2+Quantum dots; the DNA design principle is as follows: 5-20 phosphorothioate modified bases + 4-10 connecting arms + DNA sequence + modified azide or diphenyl cyclooctyne, wherein the DNA sequence plays a role in connection in one aspect and is connected with CdTe, namely Zn2+The connection quantity of the quantum dots can be regulated and controlled through the number and concentration of the phosphorothioate modified base groups; in addition, the DNA sequence has a targeting function and can react with target nucleic acid, protein, cells and the like;
2) obtained azide or diphenyl cyclooctyne modified DNA functionalized CdTe Zn2+Centrifuging and purifying the quantum dot solution by using an ultrafiltration tube with the molecular cut-off of 30KD, pouring the waste liquid, adding ultrapure water for washing and centrifuging, circularly washing and centrifuging for 4 times, and inverting and centrifuging the ultrafiltration tube to obtain azide or diphenyl cyclooctyne modified DNA functionalized CdTe, Zn2+And (4) storing the pure quantum dot product at 4 ℃ for later use.
Preferably, the DNA sequence connected to the quantum dot in the step 1) is simultaneously modified by azide or diphenyl cyclooctyne and phosphorothioate, is designed by the inventor and is biologically engineered by Shanghai biologyThe synthesis of the technical service-limited company has the nucleic acid sequences G AAAA AAA ACC TTC CTC CGC AAT ACT CCC CCA GGT AAA-N, wherein the 5 'end of the nucleic acid sequence is modified with phosphorothioate and the 3' end of the nucleic acid sequence is modified with azide or diphenyl cyclooctyne3G A AAA AAAACC TTC CTC CGC AAT ACT CCC CCA GGT AAA-DBCO (5 ' → 3 '; is modified with phosphorothioate, 3 ' end can be modified with azide or diphenyl cyclooctyne).
Preferably, in the step 1), the molar ratio of cadmium chloride, zinc chloride and N-acetyl-L-cysteine is 1 (0.5-3) to (1-10), the molar ratio of sodium tellurite and sodium borohydride is 2:1, and the concentration of cadmium chloride in the mixed aqueous solution is 0.001-0.0625 mM.
In a second aspect of the invention, the azide or diphenyl cyclooctyne modified DNA functionalized quantum dot obtained by the preparation method is provided.
In a third aspect of the invention, the application of the azide or diphenyl cyclooctyne modified DNA functionalized quantum dot in protein labeling is provided.
Preferably, the protein is glucose oxidase.
The fourth aspect of the invention provides an application of the azide or diphenyl cyclooctyne modified DNA functionalized quantum dot in cell labeling.
Preferably, the cell surface expresses a sialic acid receptor, since the added glycosyl compound will produce sialic acid through multi-step enzymatic catalysis, which can interact specifically with the cell surface sialic acid receptor.
Preferably, the cell is a HeLa cell.
The fifth aspect of the invention provides application of the azide or diphenyl cyclooctyne modified DNA functionalized quantum dot in labeling of nano materials, nucleic acids, viruses and bacteria.
The invention provides azide or diphenyl cyclooctyne modified DNA functionalized CdTe Zn2+The quantum dot is used for marking and applying glucose oxidase and HeLa cells: as shown in figure 1A, through strong interaction between sulfur and cadmium metal, the one-pot method for synthesizing azide modified DNA functionalized CdTe Zn is realized2+Quantum dots, and modifying diphenyl cyclooctyne on grapes by covalent couplingThe surface of the oxidase is treated with azide modification DNA functionalization CdTe Zn through click reaction2+The quantum dots are connected with glucose oxidase modified by diphenylcyclooctyne to obtain a compound (GOx-QDs) of the quantum dots and the glucose oxidase; GOx-QDs were then used for glucose detection. As shown in figure 1B, the diphenyl cyclooctyne modified DNA functional CdTe Zn is synthesized by a one-pot method through strong interaction between sulfur and metal cadmium2+Quantum dot prepared by adding azide-modified glycosyl compound (Ac) into HeLa cell culture solution4Mannaz), finally adding diphenyl cyclooctyne to modify DNA functional CdTe: Zn2+The quantum dots realize the high-efficiency marking and imaging of the HeLa cells.
Compared with the prior art, the invention has the advantages and beneficial effects that:
1. in the synthesis process of the quantum dots, DNA modified by azide or diphenyl cyclooctyne and thiophosphate is added, the azide or diphenyl cyclooctyne modified DNA functionalized quantum dots are synthesized by a one-step method for the first time, and the labeling reagent is applied to labeling of glucose oxidase and HeLa cells. Compared with the common covalent coupling method, the method does not need any coupling, has simple preparation method and low cost, does not need expensive facilities, and can be completed in a common laboratory.
2. Compared with other covalent coupling methods, the probe for labeling the protease has the advantages of high connection efficiency, no by-product, high labeling speed, capability of enhancing the protease activity and the stability of quantum dots and the like. The linear range of the prepared GOx-QDs for detecting glucose is 0.1-20 mu M, and the detection limit is 0.017 mu M.
3. Compared with other biological labeling methods, the probe for labeling HeLa cells has the advantages of no need of any chemical modification reagent, high labeling efficiency, high luminous efficiency and the like.
4. The DNA functionalized quantum dot modified by the azide or the diphenyl cyclooctyne can also be used for modification of other various molecules, such as self-assembly of nano materials, proteins, nucleic acids, cells, viruses, bacteria and the like, and has better universality. The quantum dot biomarker can better broaden the application of the quantum dot in the aspects of biosensing, imaging, tracing and the like.
Drawings
FIG. 1 shows azido or diphenyl cyclooctyne modified DNA functionalized CdTe Zn2+The quantum dot is used for marking glucose oxidase and HeLa cells and an application principle diagram.
FIG. 2 is the azide-modified DNA functionalized CdTe Zn synthesized in example 12+A transmission electron microscope and ultraviolet-visible light absorption spectrum characterization chart of the quantum dots.
FIG. 3 is the azide-modified DNA functionalized CdTe Zn prepared in example 12+And (3) a characterization diagram of coupling of the quantum dots and the glucose oxidase.
FIG. 3A is a circular dichroism chart before and after coupling of glucose oxidase, and FIG. 3B is a surface potential characterization result before and after coupling of glucose oxidase.
FIG. 4 is a graph of spectra and linearity of GOx-QDs prepared in example 2 for glucose detection.
FIG. 4A is a spectrum of glucose detection; fig. 4B shows that the fluorescence of the quantum dots is linearly related to the concentration of glucose in a certain concentration range.
FIG. 5 is the azide-modified DNA functionalized CdTe Zn synthesized in example 12+The quantum dots are mixed with glucose oxidase for detecting glucose.
FIG. 5A is a spectrum of glucose detection; fig. 5B shows that the fluorescence of the quantum dots is linear with the concentration of glucose over a certain concentration range.
FIG. 6 is the result of visual examination of GOx-QDs prepared in example 2 for glucose.
FIG. 6A shows the results of the visual detection of glucose using the mixture of quantum dots and glucose oxidase and two types of GOx-QDs probes, respectively; FIG. 6B is a linear relationship between the gray level of the quantum dots and the glucose concentration, and the inset shows the analysis result of GOx-QDs used in the paper chip for detecting glucose.
FIG. 7 shows the results of the measurement of blood glucose concentration using GOx-QDs prepared in example 2.
FIG. 7A is a spectrum of probes added to different blood samples; FIG. 7B shows the blood glucose concentration measured by the method and the results of measurement by a commercial instrument.
FIG. 8 shows the Diphenyl cyclooctyne modified DNA functionalized CdTe Zn synthesized in example 72+Cytotoxicity of quantum dots.
FIG. 9 shows different amounts of diphenylcyclooctyne modified DNA functionalized CdTe Zn synthesized in example 32+The quantum dots are used for HeLa cell marking and imaging results.
FIG. 10 shows the Diphenyl cyclooctyne modified DNA functionalized CdTe Zn synthesized in example 32+The different incubation times of the quantum dots and the HeLa cells are used for marking and imaging results.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way. Example 1 Azide-modified DNA functionalization CdTe Zn2+Preparation method of quantum dots
The preparation method comprises the following steps:
1) dissolving 0.025mmol of cadmium chloride, 0.025mmol of zinc chloride and 0.05mmol of N-acetyl-L-cysteine in deionized water to obtain a mixed solution A, and adjusting the pH value of the mixed solution A to 9.0 by using a sodium hydroxide solution; adding 0.01mmol of sodium tellurite and 0.005mmol of sodium borohydride into a 10mL single-mouth flask, and reacting for 5h under ice bath and anaerobic conditions to obtain a mixed solution B; quickly transferring the mixed solution B into the mixed solution A, finally adding 25nmol DNA modified by the azide and the thiophosphate together, uniformly stirring, transferring the mixture into a hydrothermal reaction kettle, and reacting for 30min at 200 ℃ to obtain the azide-modified DNA functionalized CdTe, namely Zn2+Quantum dots;
the DNA is designed and synthesized by the inventor from Shanghai, the 5 'end of the DNA is modified with phosphorothioate, and the 3' end of the DNA is modified with azide nucleic acid sequences G A AAA AAA ACC TTC CTC CGC AAT ACTCCC CCA GGT AAA-N3(5 '→ 3'; is modified with phosphorothioate, the sequence may be changed to have azide modified at the 5 'end and phosphorothioate modified at the 3' end).
2) The obtained azide modified DNA is functionalized with CdTe Zn2+Centrifuging and purifying the quantum dot solution with ultrafiltration tube with molecular cut-off of 30KD, pouring off the waste liquid, adding ultrapure water for washing and centrifuging, circularly washing and centrifuging for 4 times, and inverting and centrifuging the ultrafiltration tube to obtain the fluorescent dye modified DNA functionalized CdTe: Zn2+And (3) storing the quantum dot pure product at 4 ℃ for later use, and diluting the quantum dot pure product into an aqueous solution with the required concentration when required.
As can be seen from the transmission electron micrograph of fig. 2A, the quantum dots have good dispersibility and particle size uniformity. As can be seen from the UV-VIS spectrum of FIG. 2B, the probe has two distinct absorption peaks at 257nm and at 578nm, which are the absorption peaks of DNA and quantum dots, respectively, and thus, the azide-modified DNA has been successfully linked to the quantum dots.
Example 2 Azide-modified DNA functionalized CdTe Zn2+Method for using quantum dots for marking glucose oxidase
The marking method comprises the following steps:
first, glucose oxidase (GOx) was subjected to DBCO modification. Weighing 0.5mg of NHS-DBCO reagent, and adding 20 mu of LDMSO for dissolving; further, 10mg of GOx was weighed and dissolved in 500. mu.L of Tris-HCl buffer solution (pH 7.4,20mM), and NHS-DBCO reagent dissolved in DMSO was added to the above GOx solution and reacted overnight. And (3) carrying out centrifugal ultrafiltration on the reaction solution by using a 30KD ultrafiltration tube, wherein the ultrafiltration time is 10min, and the rotating speed is 5000 rpm. Finally, the GOx-DBCO solution obtained by ultrafiltration was added to the N prepared in example 13DNA-functionalized CdTe Zn2+Reacting in QDs solution for 4h, and performing centrifugal ultrafiltration on the reaction solution by using a 100KD ultrafiltration tube, wherein the ultrafiltration time is 10min and the rotation speed is 5000 rpm. The obtained GOx-QDs were stored in a refrigerator at 4 ℃ for further use.
FIG. 3A is a circular dichroism chart before and after coupling of glucose oxidase, as shown in the figure, the circular dichroism chart before and after modification of the glucose oxidase is obviously different, and the quantum dots are preliminarily shown to be successfully modified on the surface of the glucose oxidase. FIG. 3B shows the surface potential characterization results before and after coupling of glucose oxidase, and as shown in FIG. 3B, the surface potentials before and after modification of glucose oxidase are significantly different, further demonstrating that quantum dots are successfully modified on the surface of glucose oxidase.
Example 3A method for glucose detection by glucose oxidase-Quantum dots (GOx-QDs) complexes
The detection method comprises the following steps:
10. mu.L of GOx-QDs (400nM) prepared in example 2 were collected, and glucose was added at various concentrations to make the total volume of the reaction system 400. mu.L by adding 20mM Tris buffer (pH 8.4). The mixed solution was reacted at room temperature for 30min, and then the fluorescence spectrum was measured using 350nm as an excitation wavelength.
As shown in fig. 4A, the fluorescence of the quantum dots gradually decreased as the glucose concentration increased. As shown in FIG. 4B, in a certain concentration range, the fluorescence of the quantum dots and the concentration of glucose are in a linear relationship, the linear range is 0.1-20 μ M, and the detection limit is 0.017 μ M.
Example 4A method for glucose detection by mixing glucose oxidase with Quantum dots
The detection method comprises the following steps:
10. mu.L of the quantum dot (400nM) prepared in example 1 was added with a fixed amount of glucose oxidase (5U/mL), glucose was added at different concentrations, and finally 20mM Tris buffer (pH 8.4) was added to make the total volume of the reaction system 400. mu.L. The mixed solution was reacted at room temperature for 60min, and then the fluorescence spectrum was measured using 350nm as an excitation wavelength.
Fig. 5A is a spectrum of glucose detection, showing that the fluorescence of the quantum dots gradually decreases with increasing glucose concentration. As shown in FIG. 5B, the fluorescence of the quantum dots is linearly related to the concentration of glucose in a certain concentration range, the linear range is 1-50 μ M, and the detection limit is 0.27 μ M. Comparing the method of mixing quantum dots and glucose oxidase for glucose detection with the method of applying GOx-QDs to glucose detection [ example 3 ], GOx-QDs were found to have faster speed, wider linear range and higher detection sensitivity for glucose detection.
Example 5A method of using glucose oxidase-quantum dot complexes (GOx-QDs) for visual detection of glucose, the detection method comprising the steps of:
1) mu.L of GOx-QDs (4. mu.M) was taken, glucose was added thereto at various concentrations, and finally 20mM Tris buffer (pH 8.4) was added to make the total volume of the reaction system 200. mu.L. The mixed solution was reacted at room temperature for 10min, and then irradiated with an ultraviolet lamp having an excitation wavelength of 365nm in an ultraviolet dark box, observed and photographed.
2) mu.L of naked quantum dots (4. mu.M) were added, glucose was added at various concentrations, and 20mM Tris buffer (pH 8.4) was added to make the total volume of the reaction system 200. mu.L. The mixed solution was reacted at room temperature for 10min, and then irradiated with an ultraviolet lamp having an excitation wavelength of 365nm in an ultraviolet dark box, observed and photographed.
3) 2 μ L of GOx-QDs (400nM) was dropped on the paper chip, 2 μ L of glucose of different concentrations was added, the reaction was carried out for 2min, and then, the paper chip was irradiated with an ultraviolet lamp having an excitation wavelength of 365nM in an ultraviolet dark box, observed and photographed.
FIG. 6A shows the results of the visual detection of glucose by the mixture of quantum dots and glucose oxidase and the use of two types of GOx-QDs, respectively. The inset in FIG. 6B is the analysis result of GOx-QDs used in paper chip glucose detection, the fluorescence intensity decreases when the glucose concentration increases, and the analysis speed of the method is fast, only requiring two minutes. According to the relationship between the gray value of the quantum dot and the glucose concentration, a good linear relationship can be established.
In conclusion, the probe can realize the visual detection of the glucose and has high detection sensitivity. And the probe can also be used for paper chip analysis of glucose. The method has the advantages of high analysis speed and high sensitivity. In addition, the method can simply and quickly realize the detection of the actual sample and has certain application value in the analysis of the actual sample.
Example 6A method of using glucose oxidase-Quantum dot complexes (GOx-QDs) for blood glucose concentration determination in diabetic patients
The detection method comprises the following steps:
blood samples from normal and diabetic patients were provided by the Wuhan university Central and south Hospital. The blood sample is centrifuged to separate the plasma and obtain a serum sample, wherein the rotating speed is 5000rpm and the time is 10 min. mu.L of GOx-QDs (80nM) prepared [ example 2 ] and 1.0. mu.L of serum samples were added to Tris-HCl solution (20 mM pH 8.4) in a total volume of 400. mu.L. Reacting for 30min at room temperature, and measuring the fluorescence spectrum by taking 350nm as an excitation wavelength.
FIG. 7A is a spectrum of the probe added to different blood samples, which have different quenching effects on the fluorescence of the quantum dots, and the blood glucose concentration is calculated according to the linear relationship established in FIG. 4. Fig. 7B shows the blood glucose concentration measured by the method and the measurement result of a commercial instrument, and it can be seen that the method can accurately measure the blood glucose concentration.
Example 7A Diphenyl cyclooctyne modified DNA functionalized CdTe Zn2+Preparation method of quantum dots
The synthesis method comprises the following steps:
1) dissolving 0.025mmol of cadmium chloride, 0.025mmol of zinc chloride and 0.05mmol of N-acetyl-L-cysteine in deionized water to obtain a mixed solution A, and adjusting the pH value of the mixed solution A to 9.0 by using a sodium hydroxide solution; adding 0.01mmol of sodium tellurite and 0.005mmol of sodium borohydride into a 10mL single-mouth flask, and reacting for 5h under ice bath and anaerobic conditions to obtain a mixed solution B; quickly transferring the mixed solution B into the mixed solution A, finally adding 25nmol of DNA modified by the diphenyl cyclooctyne and the thiophosphate together, uniformly stirring, transferring the mixture into a hydrothermal reaction kettle, and reacting for 30min at 200 ℃ to obtain the diphenyl cyclooctyne modified DNA functionalized CdTe, namely Zn2+Quantum dots;
the DNA is designed by the inventor and synthesized by Shanghai, the 5 'end of the DNA is modified with phosphorothioate, the 3' end of the DNA is modified with diphenylcyclooctyne, and the nucleic acid sequences are G A AAA AAA ACC TTC CTC CGC AATACT CCC CCA GGT AAA-DBCO (5 '→ 3'; is modified with phosphorothioate, the sequences can be changed into 5 'end modified with diphenylcyclooctyne, and the 3' end of the DNA is modified with phosphorothioate).
2) The obtained CdTe: Zn2+Molecules for quantum dot solutionsCentrifuging and purifying with ultrafiltration tube with cut-off of 30KD, pouring off waste liquid, adding ultrapure water, washing and centrifuging, circularly washing and centrifuging for 4 times, and centrifuging the ultrafiltration tube upside down to obtain CdTe/Zn2+And (3) storing the pure quantum dot product at 4 ℃ for later use, and diluting the pure quantum dot product into an aqueous solution with the required concentration when required.
FIG. 8 shows the above synthesized diphenylcyclooctyne modified DNA functionalized CdTe Zn2+Cytotoxicity of quantum dots. As shown in the figure, when the concentration of the quantum dot is 250nM, the toxicity to HeLa cells is still not obvious, which indicates that the quantum dot can be better used for labeling and imaging HeLa cells.
Example 8A Diphenyl cyclooctyne modified DNA functionalized CdTe Zn2+Method for using quantum dots for HeLa cell marking
The marking method comprises the following steps:
for the diphenylcyclooctyne modified DNA prepared in example 7 functionalized CdTe Zn2The dosage of the + quantum dots is optimized, as shown in fig. 9, when more quantum dots are added, the more obvious the fluorescence intensity in the HeLa cell is; when the dosage of the quantum dots is 50 muL, the effect is equivalent to that of the quantum dots with the dosage of 40 muL, which indicates that the dosage of the quantum dots basically reaches saturation.
HeLa cells were incubated in the presence of 40. mu.M Ac4The culture medium of the Mannaz is incubated for 3, 6, 12 and 24 hours at 37 ℃. After incubation, the medium was gently blotted dry, and the cells were washed twice with 1mL PBS. Then, HeLa cells were further incubated in a medium containing DBCO-DNA functionalized quantum dots (40. mu.L) for 4h at 37 ℃. The medium containing the DBCO-DNA functionalized quantum dots was gently aspirated, and the cells were rinsed three times with 1mL of medium. HeLa cells were incubated in medium for another 30min, the medium was gently aspirated, and the cells were rinsed three times with 1mL of medium. Finally, the old medium was replaced with fresh medium and the HeLa cells were observed with a confocal microscope.
As shown in FIG. 10, the same amount of Ac was added4ManNAz, the more significant the fluorescence intensity in HeLa cells when the incubation time is longer; when the incubation time isThe imaging effect of HeLa is already very obvious at 24h, indicating that Ac4The incubation time of ManNAz was basically sufficient. The experimental results of FIGS. 9 and 10 show that diphenylcyclooctyne modified DNA functionalizes CdTe Zn2+The quantum dots can be used for non-modification and rapid marking and imaging of HeLa cells.
Sequence listing
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<120> preparation method of DNA functionalized quantum dots based on click chemistry and application of DNA functionalized quantum dots in biomarker and detection
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Claims (9)

1. The azide or diphenyl cyclooctyne modified DNA functionalized quantum dot is characterized in that the azide or diphenyl cyclooctyne modified DNA functionalized quantum dot is prepared by the following synthetic method, and specifically comprises the following steps:
1) dissolving cadmium chloride, zinc chloride and N-acetyl-L-cysteine in deionized water to obtain a mixed solution A, wherein the molar ratio of the cadmium chloride to the zinc chloride to the N-acetyl-L-cysteine is 1 (0.5-3) to 1-10, and adding sodium hydroxideThe pH value of the mixed solution A is adjusted to 8.0-9.0 by the solution, and the concentration of cadmium chloride in the mixed aqueous solution is 0.001-0.0625 mM; taking sodium tellurite and sodium borohydride in a molar ratio of 2:1, adding the mixture into a 10mL single-neck flask, placing the mixture into an ice bath and anaerobic condition for reaction for 5 hours to obtain a mixed solution B, quickly transferring the mixed solution B into the mixed solution A, finally adding 20-200 mu M DNA modified by azide or diphenylcyclooctyne and thiophosphate, uniformly stirring, transferring the mixture into a hydrothermal reaction kettle, and reacting at 150-200 ℃ for 20-35 minutes to obtain the azide or diphenylcyclooctyne modified DNA functionalized CdTe, namely Zn2+Quantum dots; the DNA modified by the azide or the diphenyl cyclooctyne and the phosphorothioate together has the nucleotide sequence G A AAA AAA ACC TTC CTC CGC AATACT CCC CCA GGT AAA-N modified by the azide or the diphenyl cyclooctyne at the 5 'end and the nucleotide sequence G A AAA AAA ACC TTC CTC CGC AATACT CCC CCA GGT AAA-N modified by the azide or the diphenyl cyclooctyne at the 3' end3G A AAA AAA ACC TTC CTC CGC AAT ACTCCC CCA GGT AAA-DBCO, phosphorothioate modification, N3Represents azido, DBCO represents diphenylcyclooctyne;
2) obtained azide or diphenyl cyclooctyne modified DNA functionalized CdTe Zn2+Centrifuging and purifying the quantum dot solution by using an ultrafiltration tube with the molecular cut-off of 30KD, pouring the waste liquid, adding ultrapure water for washing and centrifuging, circularly washing and centrifuging for 4 times, and inverting and centrifuging the ultrafiltration tube to obtain azide or diphenyl cyclooctyne modified DNA functionalized CdTe: Zn2+And (4) storing the pure quantum dot product at 4 ℃ for later use.
2. The use of the azide or diphenylcyclooctyne modified DNA functionalized quantum dot of claim 1 in protein labeling.
3. Use according to claim 2, wherein the protein is glucose oxidase.
4. The use of the diphenylcyclooctyne modified DNA functionalized quantum dot of claim 1 in cell labeling.
5. Use according to claim 4, wherein the cell surface expresses a sialic acid receptor.
6. Use according to claim 5, wherein the cells are HeLa cells.
7. The azide or diphenyl cyclooctyne modified DNA functionalized quantum dot as claimed in claim 1, and application of the quantum dot in nanometer materials, nucleic acids, viruses and bacteria labeling.
8. The glucose oxidase-quantum dot GOx-QDs compound is characterized by being prepared by the following synthetic method, and specifically comprises the following steps:
firstly, the glucose oxidase GOx was subjected to DBCO modification: weighing 0.5mg NHS-DBCO reagent, adding 20 mu L DMSO for dissolving; weighing 10mg GOx, dissolving in 500 μ L of 20mM Tris-HCl buffer solution with pH 7.4, adding NHS-DBCO reagent dissolved in DMSO into the GOx solution, and reacting overnight; carrying out centrifugal ultrafiltration on the reaction solution by using a 30KD ultrafiltration tube to obtain GOx-DBCO, wherein the ultrafiltration time is 10min, and the rotating speed is 5000 rpm; and finally, adding the GOx-DBCO solution obtained by ultrafiltration into the azide-modified DNA functionalized quantum dot solution in the claim 1, reacting for 4 hours, carrying out centrifugal ultrafiltration on the reaction solution by using a 100KD ultrafiltration tube, wherein the ultrafiltration time is 10min, the rotation speed is 5000rpm, and storing the obtained glucose oxidase-quantum dot GOx-QDs in a refrigerator at 4 ℃ for later use.
9. Use of the glucose oxidase-quantum dot GOx-QDs complex of claim 8 in the preparation of a reagent for detecting glucose.
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