CN110618112B - Preparation method and application of aptamer fluorescence sensor based on AuNPs @ ZIF-8 - Google Patents
Preparation method and application of aptamer fluorescence sensor based on AuNPs @ ZIF-8 Download PDFInfo
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Abstract
The invention discloses a preparation method and application of an aptamer fluorescence sensor based on AuNPs @ ZIF-8, ZIF-8 with the particle size of about 70nm is synthesized, gold nanoparticles are synthesized in situ by utilizing the reduction effect of sodium borohydride on chloroauric acid, sulfydryl and the gold nanoparticles are combined to form a gold-sulfur bond, sulfydryl modified FAM-S1 and locked DNAzyme are modified on the gold nanoparticles, miRNA and a locked chain are complementarily paired under the condition that target miRNA exists, DNAzyme is released, ZIF-8 is degraded to release zinc ions when the pH value is acidic, DNAzyme cuts a matrix chain S1 under the assistance of zinc ions to release FAM signals, and signal amplification is realized by the walking of DNAzyme on the surfaces of the gold nanoparticles.
Description
Technical Field
The invention belongs to the technical field of biosensors, and particularly relates to a preparation method and application of an aptamer fluorescence sensor based on AuNPs @ ZIF-8.
Background
MicroRNAs (miRNAs) are small molecule non-coding ribonucleic acids (RNAs) with about 19-25 nucleotides playing a key role in eukaryotes, and are found to serve as regulators of gene expression after transcription in eukaryotic cells, and play important regulation roles in cell migration, proliferation, apoptosis and other processes. There is increasing evidence that aberrant expression of miRNAs is associated with a number of diseases, including malignancies, diabetes and viral infections. The expression of miRNAs can be used as biomarkers for the diagnosis and treatment of cancer and other diseases. With the rapid development of basic research in embryology and oncology, miRNAs are receiving increasing attention as one of the important mechanisms of genetic epigenetics.
Many conventional detection methods have been used to detect miRNAs, including Northern hybridization, real-time PCR, catalytic hairpin amplification, and the like. However, these methods have certain limitations. Northern hybridization is complicated and requires radiolabelling, which not only causes serious contamination, but also has low sensitivity. Real-time PCR and catalytic hairpin amplification, while highly sensitive, require expensive instrumentation and reagents and complex assembly methods, thus limiting the widespread use of these detection methods. To overcome the shortcomings of these conventional methods, it is necessary to develop low-cost, high-sensitivity methods.
Therefore, there is a need to provide a simple, low detection limit and selective method for the detection of miRNAs.
Disclosure of Invention
The invention aims to provide a preparation method and application of an aptamer fluorescence sensor based on AuNPs @ ZIF-8, ZIF-8 with the particle size of about 100nm is synthesized, gold nanoparticles are synthesized in situ by utilizing the reduction effect of sodium borohydride on chloroauric acid, and sulfydryl and the gold nanoparticles are combined to form a gold-sulfur bond to modify sulfydryl modified FAM-S1 and locked DNAzyme onto the gold nanoparticles. Under the condition that a target object miRNA exists, the miRNA and the locking strand are complementarily paired, the DNAzyme is released, when the pH is acidic, ZIF-8 is degraded to release zinc ions, the DNAzyme cuts a substrate strand S1 under the assistance of the zinc ions to release FAM signals, and signal amplification is realized through the walking of the DNAzyme on the surface of the gold nanoparticles.
The invention provides a preparation method of an aptamer fluorescence sensor based on AuNPs @ ZIF-8, which comprises the following steps:
(1) preparing an AuNPs @ ZIF-8 material;
(2) mixing the DNA Lock1 solution with the DNA DNAzyme solution, heating and cooling to obtain a locked DNAzyme solution;
(3) adding the locked DNAzyme solution and the DNA S1 solution into the AuNPs @ ZIF-8 solution, adding the Tween-20 solution, incubating at room temperature for 10-15 h,
(4) adding sodium chloride aqueous solution into the step (3) for four times, wherein the time interval of each time is 40-45 min; and continuously culturing for 22-26 h after the addition is finished, centrifuging and washing the obtained product, and dispersing the product in a buffer solution to obtain the aptamer fluorescence sensor DNAzyme-AuNPs @ ZIF-8 based on AuNPs @ ZIF-8.
In the step (1), the preparation method of the AuNPs @ ZIF-8 material comprises the following steps: dropwise adding a chloroauric acid aqueous solution into the methanol solution of the ZIF-8, and stirring for reaction for 5-8 hours; and then, quickly adding a newly-configured sodium borohydride methanol solution, continuously reacting for 1-1.5 h, and cleaning, centrifuging and drying the obtained product to obtain the AuNPs @ ZIF-8 material which is of a dodecahedron structure and has an average particle size of 70 nm.
Further, the concentration of the methanol solution of the ZIF-8 is 1.5-2.5 mg/mL; the mass concentration of the chloroauric acid aqueous solution is 0.8-1.5%, the concentration of the sodium borohydride methanol solution is 6.0-8.0 mg/mL, and the volume ratio of the ZIF-8 methanol solution to the chloroauric acid aqueous solution to the sodium borohydride methanol solution is 8-12: 0.2-0.4: 1.
further, the preparation method of the ZIF-8 comprises the following steps: and slowly adding the methanol solution of zinc nitrate into the methanol solution of 2-methylimidazole, stirring for 15-20 minutes, and cleaning, centrifuging and drying the product to obtain the ZIF-8 material.
The concentration of the methanol solution of the zinc nitrate is 21.4-25.0 mg/mL; the concentration of the methanol solution of the 2-methylimidazole is 50-55 mg/mL; the volume ratio of the two is 1: 1.
The DNA Lock1 solution, the DNA DNAzyme solution, the DNA S1 solution and the AuNPs @ ZIF-8 solution are respectively obtained by dissolving 2.5OD DNA Lock1, DNA DNAzyme, DNA S1 and AuNPs @ ZIF-8 in 25mM Tris-HCl buffer solution with pH 7.4.
The concentrations of the DNA Lock1 solution, the DNA DNAzyme solution and the DNA S1 solution are all 100 mu M, and the concentration of the AuNPs @ ZIF-8 solution is 1 mg/mL.
The sequences of the DNA Lock1, the DNA DNAzyme and the DNA S1 are respectively as follows:
DNA Lock1：5'-TTGAAG CAC AAA TTC GGT TCT ACA GGG TA-3’；
DNA DNAzyme：
5'-SH-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTC TTCCGACCGGTCGAAAATAGTGGCCCGAATTTGTGCTTCAA-3’；
DNA S1：5’-SH-TTTTTTTTTTTTTTGGGCCACTAT rAGGAAT-FAM-3’。
in the step (2), the volume ratio of the DNA Lock1 solution to the DNA DNAzyme solution is 1: 3; the heating and cooling refer to: heating to 70-80 ℃, keeping for 3-5 min, and then slowly cooling to room temperature at the speed of 0.5-1.5 ℃/min.
In the step (3), the volume ratio of the locked DNAzyme solution, the DNA S1 solution and the AuNPs @ ZIF-8 solution is 1:5: 194; the final concentration of tween-20 in the system was 0.05%.
In the step (4), the concentration of the sodium chloride aqueous solution is 0.1M; the ratio of the volume of the aqueous sodium chloride solution to the volume of the DNA S1 solution added in each case was 2: 1.
In step (4), the washing is performed once with 10mM Tris-HCl buffer solution (pH7.4) containing 0.05% Tween 20.
The invention also provides application of the aptamer fluorescence sensor based on AuNPs @ ZIF-8 prepared by the preparation method in miRNA detection.
Further, the detection method of the miRNA comprises the following steps:
dispersing the aptamer fluorescence sensor based on AuNPs @ ZIF-8 into a Tris-HCl buffer solution with the pH value of 5.5, respectively adding a series of miRNA-10b solutions with different concentrations, culturing, detecting fluorescence signals corresponding to each system, and constructing a linear relation between fluorescence intensity and the concentration of the miRNA-10b solution so as to realize quantitative detection of miRNA-10 b.
Further, the culture condition is that the culture is carried out for 60-70 min at 35 ℃.
Further, the sequence of the miRNA-10b is as follows:
5'-UACCCUGUAGAACCGAAUUUGUG-3', respectively; the concentration of the miRNA-10b solution is 0.01nM-100nM respectively.
The preparation method of the miRNA-10b solution comprises the following steps: 2.5OD miRNA-10b was dissolved in DEPC water.
In the detection method, when the pH value is equal to 5.5, the DNAzyme-AuNPs @ ZIF-8 aptamer fluorescence sensor is decomposed to release zinc ions and gold nanoparticles with modified DNA, under the condition that miRNA-10b exists, miRNA-10b and DNA Lock1 are complementarily paired to release DNAzyme, under the condition that zinc ions exist, DNAzyme cuts specific base sites on matrix strand DNA DNAS1 to release DNA fragments with FAM, and therefore fluorescence recovery is achieved. A plurality of DNAzyme chains and matrix chains are modified on one gold nanoparticle, and signal amplification is realized by the running of the DNAzyme on the gold nanoparticle. The fluorescence intensity is increased along with the increase of the concentration of the miRNA, so that the sensitive detection of the miRNA is realized.
The preparation method of the aptamer fluorescence sensor based on AuNPs @ ZIF-8, which is provided by the invention, has the advantages that the synthesis by using AuNPs @ ZIF-8 is simple, the energy consumption is low, the cost is low, the biocompatibility is good, DNA and a metal organic framework are related, the pH response of ZIF-8 is utilized, and then a walking amplification system is constructed by utilizing the DNA base complementary pairing principle and the specific cutting of DNAzyme, so that the specificity and sensitivity detection of miRNA is realized.
Drawings
FIG. 1 is a schematic diagram of the synthesis process of AuNPs @ ZIF-8 and the construction of a fluorescence sensor for miRNA detection;
FIGS. 2A and 2B are Scanning Electron Micrographs (SEM) of ZIF-8 and AuNPs @ ZIF-8, respectively, FIGS. 2C and 2D are Transmission Electron Micrographs (TEM) of ZIF-8 and AuNPs @ ZIF-8, respectively, FIGS. 2E-L are elemental profiles of AuNPs @ ZIF-8 material, and FIG. 2M is a High Resolution Transmission Electron Micrograph (HRTEM) of AuNPs @ ZIF-8;
FIGS. 3A and 3B are respectively a powder X-ray diffraction Pattern (PXRD) and a Fourier transform infrared spectrum (FT-IR) of ZIF-8 and AuNPs @ ZIF-8, FIG. 3C is an ultraviolet absorption curve of ZIF-8, ZIF-8+ DNA, AuNPs @ ZIF-8+ DNA, and FIG. 3D is a Zeta potential pattern of ZIF-8, AuNPs @ ZIF-8+ DNA;
FIG. 4A is a standard curve for detecting miRNA at different concentrations based on DNAzyme-AuNPs @ ZIF-8 aptamer fluorescence sensor, and FIG. 4B is a corresponding fluorescence curve;
FIG. 5 is a diagram showing the feasibility of miRNA detection based on DNAzyme-AuNPs @ ZIF-8 aptamer fluorescence sensors, wherein A is a fluorescence curve of a target added under different pH values, and B is a fluorescence intensity curve of incubation for different times after miRNA addition;
FIG. 6 is a diagram of the optimization of miRNA detection conditions based on DNAzyme-AuNPs @ ZIF-8 aptamer fluorescence sensor, wherein A is the optimization of incubation temperature, and B is DNA S1: optimizing the proportion of DNAzyme;
FIG. 7 is a selectivity graph for detecting different miRNAs based on DNAzyme-AuNPs @ ZIF-8 aptamer fluorescence sensors, and the corresponding fluorescence curves are shown in the inset.
Detailed Description
The invention is described in detail below with reference to the following examples and the accompanying drawings.
Example 1
The preparation method of the AuNPs @ ZIF-8 material comprises the following steps:
(1) weighing 350mg of 2-methylimidazole (2-MIM), dissolving in 7mL of methanol, and dissolving for 10-15 minutes by ultrasonic wave;
(2) 150mg of zinc nitrate [ Zn (NO) was weighed3)2·6H2O]Dissolving in 7mL of methanol, and ultrasonically dissolving for 8-10 minutes;
(3) slowly adding the zinc nitrate solution obtained in the step (2) into the 2-methylimidazole solution obtained in the step (1) under the condition of stirring, continuously stirring for 20-30 minutes at room temperature, centrifuging the obtained product, washing the product with absolute methanol for three times, and drying the product in a vacuum drying oven at 80 ℃ for 12 hours to obtain a ZIF-8 material, wherein SEM and TEM images of the ZIF-8 material are shown in figures 2A and 2C, and the ZIF-8 material is of a dodecahedron structure, has the particle size of about 70nm and is uniform in appearance;
(4) weighing 20mg of ZIF-8 material, dispersing in 10mL of anhydrous methanol, performing ultrasonic treatment for 15-20 minutes, dropwise adding 300 mu L of chloroauric acid aqueous solution under stirring, continuously stirring at room temperature for 6 hours, and rapidly adding 1mL of newly prepared sodium borohydride (NaBH) containing 7.2mg4) The mixture is stirred and reacted for 1 hour at room temperature, the product is centrifuged and washed with absolute methanol for three times, and the product is dried in a vacuum drying oven for 24 hours at the temperature of 80 ℃ to obtain AuNPs @ ZIF-8 material.
SEM and TEM images are shown in figures 2B and 2D, the AuNPs @ ZIF-8 material can be seen to keep a dodecahedron structure, the particle size is about 70nm, small-particle gold nanoparticles exist on the surface, HRTEM image 2M proves successful coating of the gold nanoparticles, element distribution diagrams are shown in figures 2E-L, and successful synthesis of the AuNPs @ ZIF-8 is also proved.
Example 2
A construction process based on AuNPs @ ZIF-8 fluorescence sensor comprises the following steps:
(1) weighing 2mg of AuNPs @ ZIF-8, dissolving in 2mL of Tris-HCl buffer solution (25mM, pH 7.4), and performing ultrasonic treatment for about 20 minutes to uniformly disperse to obtain a solution containing AuNPs @ ZIF-8;
(2) the purchased DNA sequences DNA S1, DNA Lock1 and DNA DNAzyme are respectively dissolved in 25mM Tris-HCl buffer solution with the pH value of 7.4 to obtain DNA S1 solution, DNA Lock1 solution and DNA DNAzyme solution with the concentration of 100 uM; the sequences of the DNA S1, the DNA Lock1 and the DNA DNAzyme are respectively shown as follows:
DNA S1：5’-SH-TTTTTTTTTTTTTTGGGCCACTAT rAGGAAT-FAM-3’；
DNA Lock1：5'-TTGAAG CAC AAA TTC GGT TCT ACA GGG TA-3’；
DNA DNAzyme：
5'-SH-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTC TTCCGACCGGTCGAAAATAGTGGCCCGAATTTGTGCTTCAA-3’；
(3) adding 5 mu L of the DNA DNAzyme solution obtained in the step (2) into 15 mu L of the DNA Lock1 solution obtained in the step (2), heating to 75 ℃, keeping for 3-5 min, and slowly cooling to room temperature at the temperature of 1 min/DEG C to obtain a locked DNAzyme solution;
(4) adding 1 mu L of the locked DNAzyme solution obtained in the step (3) and 5 mu L of the DNA S1 solution obtained in the step (2) into 194 mu L of the AuNPs @ ZIF-8 solution obtained in the step (1), adding 1% of Tween20 to enable the mass concentration of the final Tween20 to be 0.05%, and culturing at room temperature for 12 h;
(5) adding a 0.1M sodium chloride aqueous solution into the reaction system in the step (4) in four times to increase the loading capacity of the DNA on the AuNPs @ ZIF-8, wherein the adding amount is 10 mu L each time, performing ultrasonic treatment for about 1 minute after adding an NaCl aqueous solution each time, incubating at room temperature for 40 minutes, continuing to incubate for 24 hours after adding all the DNA, centrifuging, washing three times by using a 10mM Tris-HCl slow solution with pH7.4 containing 0.05% Tween20, dispersing the obtained product in a 25mM Tris-HCl buffer solution with pH5.5, and storing at 4 ℃ for later use, namely a DNAzyme-AuNPs ZIF-8 fluorescence sensor solution.
As can be seen from the UV absorption curve in FIG. 3C, the characteristic absorption peak of AuNPs has a slight red shift and the characteristic peak of DNA appears at 260nm, which can prove the successful preparation of DNAzyme-AuNPs @ ZIF-8 fluorescence sensor, and meanwhile, the successful preparation of DNAzyme-AuNPs @ ZIF-8 fluorescence sensor can also be proved according to the Zeta potential variation diagram in FIG. 3D.
Example 3
DNAzyme-AuNPs @ ZIF-8 fluorescent sensor for quantitatively detecting miRNA-10b, wherein the sequence of miRNA-10b is 5'-UACCCUGUAGAACCGAAUUUGUG-3'
The DNAzyme-AuNPs @ ZIF-8 fluorescent sensor solution obtained in example 2 with the concentration of 1mg/mL is divided into a plurality of groups, miRNA-10b solution with the concentration of 0.01nM to 100nM (obtained by dissolving 2.5OD miRNA-10b in DEPC water) is respectively added into the groups, and the groups are cultured for 60min at 35 ℃; then, the fluorescence emission spectrum of each system is detected under the excitation wavelength of 490nm, and the result is shown in FIG. 4B;
the concentration of the miRNA-10b solution is used as an abscissa, the maximum value of the fluorescence intensity of each system is used as an ordinate, and a standard curve is constructed as shown in FIG. 4A, so that a linear equation is obtained, namely I is 346.26log [ c ]]+936.5728, its correlation coefficient R20.9720, the detection limit is 3pM, and the quantitative detection of the miRNA-10b solution with unknown concentration can be realized according to a linear curve and a linear equation.
Example 4
Feasibility study for detecting miRNA-10b based on DNAzyme-AuNPs @ ZIF-8 fluorescent sensor
(1) Adding 20 mu L of miRNA-10b solution with the concentration of 1mg/mL into the DNAzyme-AuNPs @ ZIF-8 fluorescent sensor solution obtained in the embodiment 2, culturing at 35 ℃ for 60min, and detecting the fluorescence intensity, wherein as can be seen from FIG. 5A, only when the pH is acidic, the target is added for culturing, the fluorescent signal is obtained, and the feasibility of detecting the miRNA-10b based on the DNAzyme-AuNPs @ ZIF-8 fluorescent sensor is proved;
(2) 20 mu L of 1nM miRNA-10B solution is added into the DNAzyme-AuNPs @ ZIF-8 fluorescence sensor solution with the concentration of 1mg/mL obtained in example 2, and the fluorescence intensity is detected after incubation for different time at 35 ℃, as shown in FIG. 5B, it can be seen that the fluorescence intensity is continuously increased along with the increase of time and becomes stable after 60min, and the fact that the fluorescence intensity can be amplified when the DNAzyme walks is proved.
Example 5
Detecting miRNA-10b optimization conditions based on DNAzyme-AuNPs @ ZIF-8 fluorescent sensor:
(1) adding 20 mu L of 1nM miRNA-10b solution into the DNAzyme-AuNPs @ ZIF-8 fluorescence sensor solution with the concentration of 1mg/mL obtained in the example 2, changing the incubation temperatures to be 20, 25, 30, 35, 40 and 45 ℃ respectively, and the incubation time to be 60min, detecting the corresponding fluorescence intensity, wherein the result is shown in FIG. 6A, which shows that 35 ℃ is the optimal temperature for the experiment;
(2) the volumes of the DNA S1 buffer solutions in the step (4) in example 2 were respectively replaced with 2, 3, 4, 5, 6, 7 and 8. mu.L, 20. mu.L of 1nM miRNA-10B solutions were respectively added to the DNAzyme-AuNPs @ ZIF-8 fluorescence sensor solutions obtained at the concentration of 1mg/mL, and the fluorescence intensity was measured after incubation at 35 ℃ for 60min, as shown in FIG. 6B, which indicates that the DNA S1: the fluorescence intensity of the system was maximal at a molar ratio of the locked DNAzyme of 20: 1.
Example 6
7 groups of DNAzyme-AuNPs @ ZIF-8 fluorescent sensor solutions with the concentration of 1mg/mL obtained in the example 2 are respectively added with miRNA-10b, miRNA-16, miRNA-126, single base mismatch miRNA-10b interferent, two base mismatch miRNA-10b interferent and three base mismatch-miRNA-10 b interferent solutions with the volume and concentration of 20 muL and 1nM, and the miRNA-10b interferent solutions are all solutions prepared by dissolving in DEPC water, wherein one group is a blank group, and after incubation at 35 ℃ for 60min, the fluorescence intensity obtained by each group correspondingly is detected, as shown in FIG. 7, which shows that the fluorescent sensor has good selectivity.
The above detailed description of the preparation method and application of an AuNPs @ ZIF-8-based aptamer fluorescence sensor with reference to the examples is illustrative and not restrictive, and several examples can be listed according to the limited scope, so that variations and modifications without departing from the general concept of the present invention shall fall within the scope of the present invention.
SEQUENCE LISTING
<110> university of teacher's university in Anhui
<120> preparation method and application of aptamer fluorescence sensor based on AuNPs @ ZIF-8
<130> 1
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 29
<212> DNA
<213> DNA Lock1
<400> 1
5'- ttgaagcaca aattcggttc tacagggta-3’ 29
<210> 2
<211> 89
<212> DNA
<213> DNA DNAzyme
<400> 2
5'-SH-tttttttttt tttttttttt tttttttttt tttttttttt ttttattctt ccgaccggtc 60
gaaaatagtg gcccgaattt gtgcttcaa-3’ 89
<210> 3
<211> 31
<212> DNA
<213> DNA S1
<400> 3
5’-SH-tttttttttt ttttgggcca ctatraggaa t-FAM-3’ 31
<210> 4
<211> 23
<212> RNA
<213> miRNA-10b
<400> 4
5'-uacccuguag aaccgaauuu gug-3’ 23
Claims (9)
1. A preparation method of an aptamer fluorescence sensor based on AuNPs @ ZIF-8 for quantitative detection of miRNA-10b is characterized by comprising the following steps:
(1) preparing an AuNPs @ ZIF-8 material;
(2) mixing the DNA Lock1 solution with the DNA DNAzyme solution, heating and cooling to obtain a locked DNAzyme solution;
(3) adding the locked DNAzyme solution and the DNA S1 solution into the AuNPs @ ZIF-8 solution, adding the Tween-20 solution, and culturing at room temperature for 10-15 h;
(4) adding the sodium chloride aqueous solution into the step (3) for four times, wherein the time interval of each addition is 40-45 min; continuing to cultivate for 22-26 h after the addition is finished, centrifuging and washing the obtained product, and dispersing the product in a buffer solution to obtain the aptamer fluorescence sensor DNAzyme-AuNPs @ ZIF-8 based on AuNPs @ ZIF-8;
the molar ratio of the DNA S1 to the locked DNAzyme is 20: 1;
the sequences of the DNA Lock1, the DNA DNAzyme and the DNA S1 are respectively as follows:
DNA Lock1：5'-TTGAAG CAC AAA TTC GGT TCT ACA GGG TA-3’；
DNA DNAzyme：
5'-SH-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTCTTCCGACCGGTCGAAAATAGTGGCCCGAATTTGTGCTTCAA-3’；
DNA S1：5’-SH-TTTTTTTTTTTTTTGGGCCACTAT rAGGAAT-FAM-3’。
2. the preparation method of claim 1, wherein in the step (1), the AuNPs @ ZIF-8 material is prepared by: dropwise adding a chloroauric acid aqueous solution into a methanol solution of ZIF-8, and stirring for reaction for 5-8 hours; and then quickly adding a newly configured sodium borohydride methanol solution, continuously reacting for 1-1.5 h, and cleaning, centrifuging and drying the obtained product to obtain the AuNPs @ ZIF-8 material.
3. The preparation method according to claim 1, wherein the concentration of the methanol solution of ZIF-8 is 1.5 to 2.5 mg/mL; the weight concentration of the chloroauric acid aqueous solution is 0.8-1.5%; the concentration of the sodium borohydride methanol solution is 6.0-8.0 mg/mL, and the volume ratio of the ZIF-8 methanol solution to the chloroauric acid aqueous solution to the sodium borohydride methanol solution is 8-12: 0.2-0.4: 1.
4. the method according to claim 1 or 3, wherein the concentration of the DNA Lock1 solution, the DNA DNAzyme solution, and the DNA S1 solution is 100. mu.M, and the concentration of the AuNPs @ ZIF-8 solution is 1 mg/mL.
5. The method according to claim 1 or 3, wherein in the step (2), the volume ratio of the DNA Lock1 solution to the DNA DNAzyme solution is 1: 3; the heating and cooling refer to: heating to 70-80 ℃, keeping for 3-5 min, and then slowly cooling to room temperature at the speed of 0.5-1.5 ℃/min.
6. The method according to claim 1 or 3, wherein in the step (3), the volume ratio of the locked DNAzyme solution, the DNA S1 solution and the AuNPs @ ZIF-8 solution is 1:5: 194; the final concentration of tween-20 in the system was 0.05%.
7. The production method according to claim 1 or 3, wherein in the step (4), the concentration of the aqueous sodium chloride solution is 0.1M; the volume ratio of the sodium chloride aqueous solution to the DNA S1 solution added each time was 2: 1.
8. The application of the AuNPs @ ZIF-8-based aptamer fluorescence sensor prepared by the preparation method according to any one of claims 1-7 in miRNA detection.
9. The use according to claim 8, wherein the method for detecting the miRNA comprises the following steps:
dispersing the aptamer fluorescence sensor based on AuNPs @ ZIF-8 into a Tris-HCl buffer solution with the pH value of 5.5, respectively adding a series of miRNA-10b solutions with different concentrations, culturing, detecting fluorescence signals corresponding to each system, and constructing a linear relation between fluorescence intensity and the concentration of the miRNA-10b solution, thereby realizing the quantitative detection of miRNA-10 b.
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Non-Patent Citations (5)
| Title |
|---|
| An enzyme-free and label-free fluorescence biosensor for microRNAdetection based on cascade amplification of DNAzyme-poweredthree-dimensional DNA walker and hybridization chain reaction;Wang Rui等;《Sensors and Actuators B》;20180422;全文 * |
| Au nanoparticles with enzyme-mimicking activity-ornamented ZIF-8 for highly efficient photodynamic therapy;Ma Yin-Chu等;《Biomaterials Science》;20190625;第2741页左栏第1段-第2746页右栏第2段 * |
| DNAzyme Based Nanomachine for in Situ Detection of MicroRNA in Living Cells;Liu Jing等;《ACS Senors》;20171128;第1848页左栏第1段-1852页左栏第3段 * |
| DNAzyme-Loaded Metal–Organic Frameworks (MOFs) for Self-Sufficient Gene Therapy;Wang Huimin等;《Angewandte Chemie International Edition》;20190527;第7381页左栏第1段-第7383页右栏第2段 * |
| Nanoscale Zeolitic Imidazolate Framework‑8 for Ratiometric Fluorescence Imaging of MicroRNA in Living Cells;Yi Jin-Tao等;《Analytical Chemistry》;20171121;全文 * |
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