CN110387416B - Nano-gold dual-probe system with controllable polymerization regulation function and application thereof - Google Patents

Nano-gold dual-probe system with controllable polymerization regulation function and application thereof Download PDF

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CN110387416B
CN110387416B CN201810338901.XA CN201810338901A CN110387416B CN 110387416 B CN110387416 B CN 110387416B CN 201810338901 A CN201810338901 A CN 201810338901A CN 110387416 B CN110387416 B CN 110387416B
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龙亿涛
钱若灿
吕键
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East China University of Science and Technology
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Abstract

The invention provides a nanogold double-probe system with a controllable polymerization regulation function, which comprises an even number of probes matched with each other, wherein each pair of probes is a DNA single-chain molecular beacon-odd modified nanogold probe-odd and a molecular beacon-even modified nanogold probe-even respectively, the ends of the molecular beacon-odd and the molecular beacon-even are designed into a pair of DNA single-chain segments which are matched with each other, the molecular beacon-odd and the molecular beacon-even can recognize target microRNA molecules in cells and generate configuration change, and after the recognition and configuration change processes, the ends of the molecular beacon-odd and the molecular beacon-even generate hybridization combination, so that the molecular beacon-odd and the molecular beacon-even are connected, thereby causing the aggregation of the nanogold probe-odd and the nanogold probe-even. The nanogold double-probe system with the controllable polymerization regulation and control function can effectively inhibit an extracellular secretion pathway and improve the retention time of the nanoparticles in cells.

Description

Nano-gold dual-probe system with controllable polymerization regulation function and application thereof
Technical Field
The invention relates to the fields of cell imaging analysis technology, cell pathway regulation and preparation of nano materials, in particular to a plasma nano-gold dual-probe system with a controllable polymerization regulation function and application thereof in preparation of a cell exocrine pathway inhibitor.
Background
In recent years, due to the rapid development of nanotechnology, various functionalized nanoparticles have attracted much attention, and these specifically functionalized nanoparticles are used in a large number of applications in cell research, particularly as carriers for various chemical agents to enter living cells. The plasma noble metal nanoparticles, such as gold particles with nanoscale, can effectively absorb and scatter incident light, and cause the surface plasma resonance of the gold particles through the interaction of the electron cloud on the particle surface and the incident light. Meanwhile, the plasma gold nanoparticles have stable properties, and the surfaces of the gold nanoparticles are easy to modify, so that the preparation of cell delivery systems with different biological functions is facilitated.
Currently, a variety of plasma-based cell delivery systems for gold nanoparticles have been proposed. Generally consists of a surface modification part and a core gold nanoparticle carrier. According to the specific biological functions of the surface modification part, cell targeting molecules, biological function strengthening molecules, drug molecules, functional nucleic acids, imaging reagents, photo-thermal response groups, chemical functional group carriers and the like can be modified on the surfaces of the gold nanoparticles so as to achieve the effects of cell identification and transmembrane transport.
Although the plasma-based cell delivery system of gold nanoparticles can effectively identify and enter cells, most of the nanoparticles are quickly released out of the cells through an exocrine channel of the cells after the gold nanoparticles enter the cells, the residence time in the cells is short, and conditions cannot be provided for long-time continuous cell living body research under many conditions.
Disclosure of Invention
The first purpose of the invention is to provide a nanogold dual-probe system with a controllable polymerization regulation function, which can inhibit an extracellular secretion pathway through polymerization regulation and control and improve the retention time of nanoparticles in cells.
The second purpose of the invention is to provide the application of the nano-gold dual-probe system with the controllable polymerization regulation function in the preparation of a live cell excretion inhibition carrier reagent.
The third objective of the present invention is to provide the application of the nanogold dual-probe system with the controllable polymerization regulation function in the imaging analysis.
In order to realize the first object of the invention, the invention provides a nanogold double-probe system with a controllable polymerization regulation function, the nano-gold double-probe system comprises an even number of probes which are matched with each other, each pair of probes is respectively a DNA single-chain molecular beacon-odd modified nano-gold probe-odd and a molecular beacon-even modified nano-gold probe-even, the ends of the molecular beacon-odd and the molecular beacon-even are designed into a pair of DNA single-chain segments which are matched with each other, the molecular beacon-odd and the molecular beacon-even can recognize target microRNA molecules in cells and generate configuration change, and after the recognition and configuration change processes, the ends of the molecular beacon-odd and the molecular beacon-even generate hybridization combination, so that the molecular beacon-odd and the molecular beacon-even are connected, thereby causing the aggregation of the nanogold probe-odd and the nanogold probe-even.
The odd and even numbers are only used for referring to the numbers of the double probes, one probe in the double probes is numbered with the odd number, and the other probe is numbered with the even number.
As a preferable scheme, the nanogold double-probe system comprises 2 probes which are matched with each other.
As a preferred scheme, the target microRNA molecule is microRNA-21, and the probe sequence is shown as follows:
molecular beacon-1: 5 '-HS- (CH2)6-CCGTTCTA TCA ACA TCA GTC TGA TAA GCT A TAGAACGG-Cy 5-3';
molecular beacon-2: 5 '-HS- (CH2)6-TAGAACGG TCA ACA TCA GTC TGA TAA GCT A CCGTTCTA-Cy 5-3'.
As a preferable scheme, the particle size of the nano gold is 10-70 nm.
As a preferable scheme, the target microRNA molecules in the cells comprise one or more of microRNA-21, microRNA-TK1, microRNA-survivin, microRNA-c-myc, microRNA-GalNAc-T, microRNA-144, microRNA-124-3p, microRNA-9-3p and microRNA-196b-5 p.
Preferably, the molecular beacon is modified with a fluorescent molecule.
Preferably, the fluorescent molecule is one of Cy5, Cy3, FITC, Alexa Fluor 488 and Alexa Fluor 405.
In order to realize the second purpose of the invention, the invention provides the application of the nano-gold dual-probe system with the controllable polymerization regulation function in the preparation of a living cell exocrine inhibition carrier reagent.
In order to realize the third purpose of the invention, the invention provides the application of the nano-gold dual-probe system with the controllable polymerization regulation function in imaging analysis.
As a preferred scheme, the nanogold double-probe system with the controllable polymerization regulation function is applied to imaging analysis, and when the particle size of nanogold is 50-70nm, fluorescent molecules are not modified on a molecular beacon; when the grain diameter of the nano gold is 10-49nm, fluorescent molecules are modified on the molecular beacon.
After the nano system enters cells, the special DNA single-chain molecular beacon modified on the surface of the double-probe system can specifically recognize microRNA and generate DNA chain configuration change, so that the aggregation of nano gold particles forming the double-probe system is triggered to form a massive aggregate, and an excretion passage is inhibited. The difference of the polymerization degree of the nanogold caused by different concentrations of the microRNA can be identified through dark field scattering spectroscopy. The distribution condition and the retention time of the nanogold aggregates in the cells can be represented by continuously observing under a dark-field microscope, and further degree information of nanogold polymerization inhibiting the extracellular secretion pathway is obtained.
Meanwhile, the configuration change of the DNA chain on the surface of the nano-gold caused by the microRNA can release the fluorescence of Cy5 fluorescein connected with the tail end of the DNA chain, and the fluorescence signal intensity of the fluorescence signal is in linear positive correlation with the concentration of the microRNA in the cell. Therefore, a fluorescence microscope can be used for obtaining a cell fluorescence image, and the distribution of microRNA in cells is analyzed and characterized.
The range of the gold nanoparticles can be 10-70nm, wherein when the gold nanoparticles are 10-49nm, fluorescent molecules (Cy5, Cy3, FITC, Alexa Fluor 488, Alexa Fluor 405 and the like) are required to be modified on a molecular beacon for achieving the purpose of imaging and used as fluorescent imaging markers; wherein, when the particle size is 50-70nm, the dark field imaging effect can be achieved by utilizing the scattered light of gold without modifying fluorescent molecules.
The invention has the advantages that: (1) aiming at the problem that the traditional nano cell carrier system has too short residence time in cells and is difficult to realize long-time continuous cell living body research, the method provides a nano gold dual-probe system with a controllable polymerization regulation function, and improves the residence time of nano particles in the cells. (2) The invention adopts the plasma nano-gold particle dark field and fluorescence imaging analysis technology, and utilizes a multichannel microscope to carry out in-situ imaging on cells, thereby being capable of carrying out visual detection on the endocytosis and exocytosis processes of nano-particles in single cells. (3) The nanogold double-probe system designed by the invention can perform in-situ imaging analysis and monitoring on the distribution of microRNA in cells under the action of a medicament, and provides a new method for researching an exocrine channel inhibition process caused by the aggregation behavior of the intracellular nanocarriers participating in regulation.
Drawings
FIG. 1 is a schematic diagram of the preparation of a nanogold dual-probe system with a controllable polymerization regulation function and its operation (taking a pair of probes as an example).
FIG. 2 is a micrograph (scale: 10 μm) of breast cancer cells treated with a concentration of anti-microRNA-21(0, 200nM) after 24 hours incubation with the dual probe system.
FIG. 3 is a microscopic image (scale: 50nM) before and after reaction of a certain concentration of microRNA-21(150nM) with a dual probe system.
FIG. 4 is an ultraviolet spectrum before and after a certain concentration of microRNA-21(150nM) reacts with a dual-probe system (a is before reaction, b is after reaction).
The reference numerals in fig. 1 are respectively:
1. 1, a nanogold probe; 2. A nanogold probe-2; 3. Fluorescein;
4. molecular beacon-1; 5. Molecular beacon-2; 6. A microRNA;
7. a nanogold aggregate; 8. A cell membrane.
Detailed Description
Hereinafter, the technique of the present invention will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for the purpose of assisting those skilled in the art in understanding the present invention, and is not intended to limit the present invention.
Example 1 Synthesis of Nanogold Dual Probe System with controlled polymerization Regulation
(1) According to the existing literature, 13nm gold colloid is synthesized as a preliminary gold seed. 0.5mL of HAuCl4(1%) the solution was added to a round bottom flask, diluted to 50mL, then heated in an oil bath until the solution boiled, after which 5mL of sodium citrate (38.8mM) solution was quickly added dropwise and the reaction continued for half an hour with constant stirring, at which time the solution was red and the resulting 13nm prepared gold seed was used for the next synthesis of gold nanoparticles 60nm in size.
(2) Preparing 60nm of nano gold based on 13nm prepared gold. Adding 100 mu L of NH2OH HCl (0.2M) solution was mixed with 1mL of the prepared gold seed solution, diluted to 25mL, and then 3.0mL of HAuCl was added dropwise slowly4(0.01%) solution, mixed and stirred for half an hour, at which time the solution was dark red. The resulting solution was centrifuged, washed and stored at 4 ℃.
(3) The nano-gold dual-probe system with the controllable polymerization regulation function is prepared based on 60nm nano-gold. Adding 10 mu L of Cy5 fluorescein labeled DNA single-stranded molecular beacon solution (100 mu M) into 1mL of nano-gold solution, stirring at room temperature for reaction overnight, and then dropwise and slowly adding PBS buffer solution (0.1mL) containing 2M NaCl into the solution to obtain the nano-gold (molecular beacon-1 modified nano-gold probe-1 and molecular beacon-2 modified nano-gold probe-2) with the functionalized molecular beacon. The above solution was centrifuged and washed twice with PBS buffer, and then redispersed in 1mL PBS. And mixing the two types of nano-gold probes in equal proportion to obtain the nano-gold dual-probe system with the controllable polymerization regulation function. The molecular beacon-1 and the molecular beacon-2 can recognize microRNA molecules in cells and generate configuration changes. After the process of recognition and configuration change, the ends of the molecular beacon-1 and the molecular beacon-2 are a pair of DNA single-chain segments matched with each other, and hybridization combination can be generated, so that the molecular beacon-1 is connected with the molecular beacon-2, and the nanogold probe-1 and the nanogold probe-2 are aggregated. Therefore, the nanogold double-probe system can be specifically aggregated after meeting target microRNA molecules in cells to form massive aggregates, so that an exocrine channel is inhibited. The specific sequence (sequence configuration designed based on microRNA-21 and containing Cy5 fluorescent label) of the double-probe surface modified DNA single strand is as follows:
molecular beacon-1: 5' -HS- (CH)2)6-CCGTTCTA TCA ACA TCA GTC TGA TAA GCT A TAGAACGG-Cy5-3’；(SEQ ID NO:1)
Molecular beacon-2: 5' -HS- (CH)2)6-TAGAACGG TCA ACA TCA GTC TGA TAA GCT A CCGTTCTA-Cy5-3’。(SEQ ID NO:2)
In the above situation, taking a pair of probes (i.e. a dual-probe system) as an example, a pair of gold nanoprobes is prepared, and both the molecular beacon-1 modified on the surface of the gold nanoprobe-1 and the molecular beacon-2 modified on the surface of the gold nanoprobe-2 can recognize a certain microRNA and change the configuration (in this example, the microRNA-21 is adopted), so that the two molecular beacons with changed configurations can be hybridized and combined, and the two gold nanoprobes are aggregated, and therefore, the gold nanoprobes controlled by the microRNA can be polymerized. The polymerization system can be used as an inhibitor of an extracellular secretion pathway. Similarly, if multiple pairs of probes (multiple sets of dual probes) are used, and each pair of probes recognizes different micrornas, the dual probes in each set can aggregate under the action of the corresponding recognized micrornas. Therefore, the invention can be generalized to the scope of multiple pairs of two-probe systems. For example, four groups of double probes capable of identifying four microRNAs, namely microRNA-TK1, microRNA-survivin, microRNA-c-myc and microRNA-GalNAc-T, and a specific sequence of eight needle surface modified DNA single strands (based on the sequence configuration designed by the microRNA-TK1, the microRNA-survivin, the microRNA-c-myc and the microRNA-GalNAc-T) are as follows:
molecular beacon-3: 5' -HS- (CH)2)6-CCGTTCTA CCAGGGAGAACAGAAACTAGAACGG-Cy5-3’；(SEQ ID NO:3)
Molecular beacon-4: 5' -HS- (CH)2)6-TAGAACGG CCAGGGAGAACAGAAACCCGTTCTA-Cy5-3’。(SEQ ID NO:4)
Molecular beacon-5: 5' -HS- (CH)2)6-CCGTTCTA TAGAGATGCGGTGGTCTAGAACGG-Cy5-3’；(SEQ ID NO:5)
Molecular beacon-6: 5' -HS- (CH)2)6-TAGAACGG TAGAGATGCGGTGGTCCCGTTCTA-Cy5-3’。(SEQ ID NO:6)
Molecular beacon-7: 5' -HS- (CH)2)6-CCGTTCTA GTGAAGCTAACGTTGAGTAGAACGG-Cy5-3’；(SEQ ID NO:7)
Molecular beacon-8: 5' -HS- (CH)2)6-TAGAACGG GTGAAGCTAACGTTGAGCCGTTCTA-Cy5-3’。(SEQ ID NO:8)
Molecular beacon-9: 5' -HS- (CH)2)6-CCGTTCTA CTTATGCGGATAGTGAATAGAACGG-Cy5-3’；(SEQ ID NO:9)
Molecular beacon-10: 5' -HS- (CH)2)6-TAGAACGG CTTATGCGGATAGTGAACCGTTCTA-Cy5-3’。(SEQ ID NO:10)
Example 2 monitoring the process of inhibiting the exocrine pathway caused by the aggregation behavior of the intracellular microRNA-21 regulated nano-carrier by using a dual-probe system
Using the MCF-7 breast cancer cell line as a model, MCF-7 cells (1.0mL, 1X 10) were first cultured6mL-1) After 24 hours of culture in a petri dish, a portion of the cells were treated with anti-microRNA-21(200 nM). Thereafter, 40. mu.L of the nanogold double-probe solution (20. mu.L of nanogold probe-1 and 20. mu.L of nanogold probe-2) was added to the petri dish and incubated at 37 ℃. After 6 hours, the cells were washed 3 times with PBS, and then immersed by adding 0.5mL of PBS until 24 hours after the addition of the probe, the cells were observed under a dark field/fluorescence microscope. Cell imaging analysis see figure 2. The color of the nanogold in the MCF-7 tumor cells which are not treated by the anti-microRNA-21 is orange red, and the Cy5 fluorescent signal is strong, which indicates that the nanogold is aggregated due to the high content of the microRNA-21. The nanogold is gathered in cells, and no obvious excretion phenomenon occurs, which indicates that the gathering of the nanogold inhibits an excretion channel. In the anti-microRNA-21 treated MCF-7 cells, the color of the nanogold is green, no obvious aggregation is caused, and no Cy5 fluorescent signal exists, which indicates that the content of the microRNA-21 is low. A part of nanogold appears outside cells, and the fact that an exocrine channel is not inhibited is proved.
Example 3 implementation of microcarrier aggregation for microRNA-21 regulation in vitro solution using a two-probe system
1.0mL of the nanogold double-probe solution (0.5mL of nanogold probe-1 and 0.5mL of nanogold probe-2) was mixed and then incubated with microRNA-21 (final concentration of 150nM) at room temperature. After 30 minutes, the reaction product was observed under a projection electron microscope. Imaging analysis see figure 3. When the nano-gold probe is not treated by microRNA-21, the nano-gold probe has good dispersibility; after the treatment of the microRNA-21, the double-probe system is subjected to large-scale aggregation in the solution to form a larger cluster. Therefore, the double-probe system can also realize the aggregation of the nano-carrier regulated and controlled by the microRNA in an in vitro solution.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, many modifications and adaptations can be made without departing from the principle of the present invention, and such modifications and adaptations should also be considered as the scope of the present invention.
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Claims (9)

1. The nanogold dual-probe system with the controllable polymerization regulation function is used for preparing a living cell exocrine inhibition carrier reagent, and comprises an even number of probes matched with each other, each pair of probes are respectively a DNA single-chain molecular beacon-odd modified nanogold probe-odd and molecular beacon-even modified nanogold probe-even, the tail ends of the molecular beacon-odd and molecular beacon-even are designed into a pair of DNA single-chain fragments matched with each other, the molecular beacon-odd and molecular beacon-even can recognize target microRNA molecules in cells and generate configuration change, and after the recognition and configuration change processes, the tail ends of the molecular beacon-odd and molecular beacon-even generate hybridization combination to enable the molecular beacon-odd and the molecular beacon-even to be connected, thereby causing the aggregation of the nanogold probe-odd and nanogold probe-even;
target microRNA molecules in the cells comprise one or more of microRNA-TK1, microRNA-survivin, microRNA-c-myc and microRNA-GalNAc-T.
2. The nanogold double-probe system with the controlled polymerization regulation function according to claim 1, wherein the nanogold double-probe system comprises 2 probes which are paired with each other.
3. The nanogold double-probe system with the controllable polymerization regulation function according to claim 2, wherein the target microRNA molecule is microRNA-TK1, and the probe sequence is as follows:
molecular beacon-3: 5' -HS- (CH)2)6-CCGTTCTA CCAGGGAGAACAGAAACTAGAACGG-Cy5-3’；
Molecular beacon-4: 5' -HS- (CH)2)6-TAGAACGG CCAGGGAGAACAGAAACCCGTTCTA-Cy5-3’。
4. The nanogold double-probe system with the controllable polymerization regulating function according to claim 1, wherein the particle size of the nanogold is 10-70 nm.
5. The nanogold double-probe system with the controlled polymerization regulation function according to claim 1, wherein a fluorescent molecule is modified on the molecular beacon.
6. The nanogold double-probe system with the controlled polymerization regulation function according to claim 4, wherein the fluorescent molecule is one of Cy5, Cy3, FITC, Alexa Fluor 488 and Alexa Fluor 405.
7. The use of the nanogold double-probe system with controllable polymerization regulation function according to any one of claims 1 to 4 in the preparation of a live cell exocrine inhibition carrier reagent.
8. The use of the nanogold double-probe system with controllable polymerization regulation function according to any one of claims 1 to 6 in imaging analysis.
9. The application of the nanogold double-probe system with the controllable polymerization regulation and control function in imaging analysis according to claim 8, wherein when the particle size of the nanogold is 50-70nm, fluorescent molecules are not modified on a molecular beacon; when the grain diameter of the nano gold is 10-49nm, fluorescent molecules are modified on the molecular beacon.
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