CN111781180A - DNA molecule machine for detecting exosome - Google Patents
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- CN111781180A CN111781180A CN202010655609.8A CN202010655609A CN111781180A CN 111781180 A CN111781180 A CN 111781180A CN 202010655609 A CN202010655609 A CN 202010655609A CN 111781180 A CN111781180 A CN 111781180A
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Abstract
The invention provides a DNA molecular machine for detecting exosome, which is formed by combining a characteristic Substrate chain and an Aptamer locked Motor chain on the surface of Gold Nanoparticles (GNP), and is used for detecting a specific biomarker in exosome by fluorescence so as to determine the concentration of exosome. According to the invention, the locking of a Motor chain is released through the specific recognition of the CD63 protein on an exosome membrane and the CD63aptamer used for locking the Motor chain on a DNA molecular machine, the Motor chain is combined with a Substrate chain, the Substrate chain can be cut off by a restriction endonuclease, the DNA molecular machine starts to operate, and a fluorescent molecule is released. The DNA molecular machine provided by the invention has high specificity and sensitivity and low cost; the CD63aptamer can be replaced by aptamers of other specific biomarkers, and the detection range of a DNA molecular machine is expanded.
Description
Technical Field
The invention belongs to the fields of biotechnology and medical diagnosis, and particularly relates to a DNA molecular machine for detecting exosomes based on Gold Nanoparticles (GNP).
Background
Cancer is an extremely dangerous disease, and it can be said that cancer and death are only one step in late stages. In the early stage of cancer, the cancer has little harm to human bodies, but tumor cells can release certain substances to induce angiogenesis; once vascularized, tumors begin to grow and metastasize at an alarming rate, ultimately leading to death, and cancer detection and diagnosis is therefore of paramount importance.
The currently used cancer diagnosis methods include Computed Tomography (CT), Positron Emission Tomography (PET), B-mode ultrasound, Magnetic Resonance Imaging (MRI), and tissue biopsy, which have been applied to cancer diagnosis, and have disadvantages such as irreversible damage to tissues caused by high-energy radiation, susceptibility to interference from human factors, small application range, high price, inability of early screening, and great trauma or risk.
The value of exosomes as tumor markers for cancer diagnosis has been highlighted. Exosomes are 50-150nm extracellular vesicles secreted by most cells, including tumor cells. Exosomes secreted by tumor cells carry large amounts of genetic material, such as DNA, RNA, proteins, etc., from the parental cells and are involved in biological functions such as intercellular communication, signal transduction, genetic material transport, and immune responses. Most of recent studies show that exosomes are closely related to tumor progression, angiogenesis and metastasis, so that the exosomes have important roles as potential tumor markers and prognostic factors, and a powerful non-invasive method is provided for the development of tumor detection. Currently, the more common techniques for detecting exosome surface markers or content are immunoblotting, mass spectrometry and flow cytometry. Since proteins cannot be amplified, many important low-concentration proteins cannot be detected by the existing detection means, and protein detection needs to be hopefully developed by a highly sensitive detection method.
It is known from a review of the literature that there is a synthetic DNA molecular machine that mimics the function of the protein motor, and that the remarkable specificity and predictability of Watson-Crick base pairing makes DNA a highly popular material for constructing synthetic molecular machine systems. However, there are still many limitations on the specific response and autonomous energy supply of DNA molecular machinery. Therefore, it is urgent to design a DNA molecular machine with simple operation, sensitive detection and low cost.
Disclosure of Invention
The technical problem to be solved by the invention is to construct a DNA molecular machine capable of detecting the exosome concentration, which has high specificity and sensitivity and low cost.
In order to solve the above technical problems, embodiments of the present invention provide a DNA molecular machine for detecting exosomes, which is composed of a feature Substrate chain and an Aptamer-locked Motor chain combined on the surface of GNP.
Wherein, one end of the Substrate chain is sulfhydryl (-SH), and the other end is Fluorescein (FAM).
Wherein the characteristic sequence of the Substrate chain is 5 '→ 3':
FAM-CACCTCAGCACTTTTTTTTTTTTTT-SH。
wherein the characteristic sequence of the Motor chain is 5 '→ 3':
GTGCTGAGGTGTAGCATTAGTGTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-SH。
wherein the size of the GNP is 13-25 nm.
Wherein the ratio of the Substrate chain to the Motor chain on the GNP surface is 5: 1-50: 1.
Wherein, the running energy of the DNA molecule is from the cutting of the specific sequence by the restriction endonuclease Nt.
The technical scheme of the invention has the following beneficial effects:
1. the DNA molecular machine provided by the invention is formed by combining a characteristic Substrate chain and an Aptamer locked Motor chain on the surface of GNP, is used for detecting a specific biomarker in an exosome by fluorescence, and further determines the concentration of the exosome. According to the invention, the locking of a Motor chain is released through the specific recognition of the CD63 protein on an exosome membrane and the CD63aptamer used for locking the Motor chain on a DNA molecular machine, the Motor chain is combined with a Substrate chain, the Substrate chain can be cut off by a restriction endonuclease, the DNA molecular machine starts to operate, and a fluorescent molecule is released.
2. The molecular machine provided by the invention has high specificity and sensitivity and low cost; the CD63aptamer can be replaced by an aptamer of other specific biomarkers, and the detection range of a DNA molecular machine is expanded.
Drawings
FIG. 1 is an electron micrograph of exosomes collected in the first embodiment of the present invention;
FIG. 2 is an electron micrograph of GNP prepared in example two;
FIG. 3 is a UV spectrum of GNP linked to a Substrate containing a FAM fluorophore;
FIG. 4 is a fluorescence spectrum of a DNA molecular machine operating under different conditions;
FIG. 5 is a fluorescent map of the operation of a DNA molecular machine at different times;
FIG. 6 is a fluorescent plot of the DNA molecular machine operating at different temperatures;
FIG. 7 is a fluorescence spectrum of a DNA molecular machine operating at different enzyme concentrations;
FIG. 8 is a fluorescent map of the DNA molecular machine response to exosomes of varying concentrations;
FIG. 9 is a standard curve of exosome concentration versus fluorescence intensity;
FIG. 10 is a fluorescent map of the DNA molecular machinery in response to exosomes from different cell line sources;
FIG. 11 is a fluorescence map of the DNA molecular machine run in different concentrations of serum;
FIG. 12 is a schematic diagram showing the construction of the DNA molecular machine of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.
The invention provides a DNA molecular machine for detecting exosome, which is formed by combining a characteristic Substrate chain and an Aptamer locked Motor chain on the surface of GNP (as shown in figure 12), wherein the size of the GNP is 13-25 nm, and the ratio of the Substrate chain to the Motor chain on the surface of the GNP is 5: 1-50: 1.
Wherein, one end of the Substrate chain is sulfhydryl (-SH), and the other end is Fluorescein (FAM). The Substrate chain characteristic sequence is 5 '→ 3':
FAM-CACCTCAGCACTTTTTTTTTTTTTT-SH。
the characteristic sequence of the Motor chain is 5 '→ 3':
GTGCTGAGGTGTAGCATTAGTGTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-SH。
in the present invention, the DNA molecule operating energy comes from the cleavage of the specific sequence by the restriction endonuclease nt.
The DNA molecular machine provided by the invention is used for detecting specific biomarkers on the surface or in the exosome, and further a method for determining the exosome concentration is used for overcoming various problems in the existing exosome detection technology. The construction method of the DNA molecular machine is simple and convenient to operate, sensitive in detection and low in cost, and the DNA molecular machine constructed on the GNP is used for specifically responding to the biomarkers contained in the exosomes, so that the exosome concentration is determined and the DNA molecular machine can be used for diagnosis and prognosis of tumors.
According to the invention, a Substrate chain (Substrate) containing FAM fluorescent label is linked on the surface of GNP through a sulfydryl (-SH) at the other end, at the time, the fluorescence on the Substrate is quenched, and meanwhile, a Motor chain (Motor) locked by a biomarker aptamer (taking CD63aptamer as an example) is linked, the biomarker on an exosome can be specifically combined with the biomarker aptamer on a DNA molecular machine, the Motor can be released and hybridized with the Substrate to form a double-chain region and activate an enzyme cutting site on the Substrate, at the time, the Substrate can be cut into two regions by adding endonuclease, the FAM-containing region is far away from the GNP to release fluorescence, the Motor continues to be hybridized with the next Substrate, and the endonuclease continues to exert an effect, so that the autonomous walking of the DNA molecular machine can be used for sensitively detecting the concentration of a specific biomarker, and further determining the concentration of the exosome.
The technical scheme of the invention is further illustrated by the following specific examples.
Example 1: cell culture and exosome collection
MCF-7, HepG2 and HeLa cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin and 100. mu.g/mL streptomycin, and then placed in a cell culture chamber (37 ℃ moist environment, 5% CO)2) And (4) medium proliferation. When the cell coverage area reaches about 80-90%, the 1640 complete culture medium in the cell culture dish (150mm) is sucked out, washed twice with a small amount of sterile PBS (PH7.4), added with a certain amount of 1640 basic culture medium, and placed in a cell culture box for incubation. And (4) sucking out the total amount of liquid in the culture dish after 48 hours, and separating exosomes from various tumor cell lines from a 1640 basic culture medium according to an ultracentrifugation method. Briefly, cells, dead cells and cell supernatantsThe pellet was removed by centrifugation at 300g, 4 ℃ for 10min, 1000g, 4 ℃ for 10min, and 10000g, 4 ℃ for 30min, respectively, and the supernatant was collected. Then, the supernatant was ultracentrifuged at 100000g for 70min at 4 ℃ to remove the supernatant; the pellet containing the exosomes was resuspended in a quantity of sterile PBS (pH7.4) and ultracentrifuged again at 100000g for 70min at 4 ℃. Finally, the supernatant was removed, and the exosome pellet was resuspended in sterile PBS (200 μ L, ph7.4) and stored at-80 ℃ until use. Exosome concentrations were determined by NTA and their morphology was observed by TEM.
The transmission electron micrograph of the exosome collected in example 1 is shown in fig. 1, the appearance of the exosome is cup-dish-shaped, the particle size is about 140nm, and the exosome is consistent with most literature reports.
Example 2: construction of DNA molecular machinery
The DNA molecular machine is based on GNPs with a diameter of 15nm, and the Substrate and the Motor chain locked by CD63aptamer are attached to the GNP surface through-SH at one end of the chain. To synthesize 15nm GNP, 100mg of HAuCl was added4·3H2O was dissolved in 300mL of deionized water and the solution was heated to boiling. 264mg of sodium citrate was dissolved in 30mL of water and added quickly to the mixture. The color of the solution changed from yellow to blue and then to blush within 5min and kept under vigorous stirring at boiling temperature for 10min, after cooling to room temperature, the GNP solution was obtained.
To completely lock the Motor strand, 1 μ L of a biotin (1mM) and 2 μ L of a CD63aptamer (1mM) were mixed in 97 μ L of PBS (pH7.4) and annealed. The mixture was heated to 95 ℃ and gradually cooled to 4 ℃ over 30 min. Then, 18 μ LSubstrate and 2 μ ltcepp (30mM) were added to the mixture and vortexed. After standing at room temperature for 20min, the mixture was added to 1mL GNP solution (3nM) and stirred slowly at room temperature for 12h before adding 10 μ L tween 20 (20%) to avoid GNP aggregation. To increase the loading of the Motor chains and superstrate, the salification was performed by adding NaCl solution to the above solution 8 times (60 μ L of 0.05m NaCl solution was used for the first two times and 30 μ L of 0.1m NaCl solution was used for the second six times) with 40min intervals. After the last addition, the mixture was allowed to stir slowly at room temperature for 24 h. The solution was then centrifuged at 16000g for 15min to remove chains that failed to attach to GNPs. After washing twice with PBS (pH7.4), it was redispersed in an amount of PBS (pH7.4) to ensure a final concentration of 3nM, to give a DNA molecular machine solution, which was stored at 4 ℃ until use, while measuring the UV spectra of Substrate, GNP and DNA using a UV spectrophotometer.
The transmission electron micrograph of the GNP prepared in example 2 is shown in FIG. 2, and the particle size is about 15 nm.
The UV spectra of the Substrate, GNP and DNA molecular machinery are shown in FIG. 3, and the absorbance of the DNA molecular machinery is increased after the Substrate is linked to GNP.
Example 3: determining the upload amount of Substrate on GNP
To determine the number of Substrate chains per GNP, Au-S was cleaved using 2-mercaptoethanol to dissociate the conjugated Substrate from the GNP surface. Briefly, the DNA nanomachine (40 μ L) was diluted to 200 μ L with PBS and 2 μ L2-mercaptoethanol was added, placed on a shaker overnight protected from light, the mixture was centrifuged and the supernatant containing the released substrate was measured by fluorescence spectroscopy. The fluorescence intensity was converted to the molar concentration of the substrate strand according to the standard curve. The average number of substrates per particle was obtained by dividing the measured molar concentration of substrate by the original GNP concentration. In this way, the number of Substrate on GNPs was estimated to be 136 per particle. The molar ratio of substrate chain to Motor chain in the conjugation reaction was 1: 18. Thus, approximately 8 motors were conjugated to each GNP.
Example 4: evaluation of operating efficiency of DNA nanomachines
The efficiency of the DNA molecular machine was evaluated in different experimental conditions by adding the DNA molecular machine (1), the mixture of DNA molecular machine and nt.bbvci nickase (2), the mixture of DNA molecular machine and exosome (3), and the mixture of DNA molecular machine and nt.bbvci nickase and exosome (4) to 180 μ L1 × Buffer (ph7.4) to 40 μ L molecular machine, adding 25 μ L exosome solution in different groups, incubating at 37 ℃, adding 2 μ L of DNA.
In addition, the change in fluorescence intensity over time for the DNA nanomachines was also evaluated. Fluorescence spectra of PBS solutions containing DNA nanomachines and nt. bbvcci nickase and exosomes were measured at different time intervals from 0s to 4h see figure 5, indicating that fluorescence is stronger the longer the DNA molecule 1 machine runs.
To evaluate the effect of incubation temperature, fluorescence was measured after incubating the sample solution in water baths at different temperatures (27 ℃, 32 ℃, 37 ℃, 42 ℃ and 47 ℃) for 4h throughout the test, as shown in FIG. 6, indicating that 37 ℃ is the optimal operating temperature for the DNA molecular machine. To evaluate the effect of enzyme concentration, different volumes of enzyme (0.125. mu.L, 0.25. mu.L, 0.5. mu.L, 1. mu.L and 2. mu.L) were added to the sample solution as shown in FIG. 7, and it can be seen that the higher the concentration of enzyme, the stronger the fluorescence.
Example 5: detection of exosomes from different cell lines
To assess the performance of DNA nanomachines in response to exosomes, fluorescence spectra of DNA molecular machines operating in response to different concentrations of exosomes were measured at specific time points. Unless otherwise stated, the sample solution contained DNA nanomachines (40 μ L), nt.bbvci (1 μ L) and exosomes at the indicated concentrations in 180 μ L1 × Buffer (ph 7.4). The corresponding fluorescence spectrum recorded at 480nm excitation wavelength is shown in fig. 8, and it can be concluded that the higher the exosome concentration is, the stronger the fluorescence it produces at a given time under the same conditions, and a standard curve is shown in fig. 9 based on its fluorescence spectrum. Meanwhile, in order to compare the expression levels of CD63 protein on the exosome membranes secreted by different cell lines, to compare the expression levels of CD63 protein on the exosome membranes secreted by different cell lines, MCF-7, HepG2 and HeLa secreted exosomes were added to the sample solution respectively during the whole test process, and then the fluorescence intensity of the sample solution was measured to obtain fig. 10, from which it can be seen that the expression levels of CD63 protein in different cell lines: HepG2> MCF-7> HeLa.
Example 6: serum anti-interference detection
In order to confirm whether the serum has an influence on the operation of the DNA molecular machine, the DNA molecular machine is respectively placed in the solution containing 10%, 20%, 30% and 40% fetal bovine serum for operation, and the fluorescence intensity is measured after 4 hours to obtain a figure 11, which shows that the serum has no influence on the operation of the DNA molecular machine.
The invention synthesizes a DNA molecular machine for detecting exosome by imitating a protein molecular machine, and the molecular machine has high specificity and sensitivity and low cost; the CD63aptamer used for locking a Motor chain on a DNA molecular machine can specifically recognize the CD63 protein on an exosome membrane, and the CD63aptamer can be replaced by aptamers of other specific biomarkers, so that the detection range of the DNA molecular machine is expanded.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. A DNA molecular machine for detecting exosomes, characterized in that it is composed of characteristic Substrate chains and Aptamer locked Motor chains bound to the GNP surface.
2. The DNA molecular machine for the detection of exosomes according to claim 1, characterized in that the Substrate chain is thiol (-SH) at one end and Fluorescein (FAM) at the other end.
3. A DNA molecular machine for the detection of exosomes according to claim 1 or 2, characterized in that the Substrate chain signature sequence is 5 '→ 3':
FAM-CACCTCAGCACTTTTTTTTTTTTTT-SH。
4. the DNA molecular machine for detecting exosomes according to claim 1, characterized in that the characteristic sequence of the Motor strand is 5 '→ 3':
GTGCTGAGGTGTAGCATTAGTGTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-SH。
5. the DNA molecular machine for detecting exosomes according to claim 1, characterized in that GNPs are 13-25 nm in size.
6. The DNA molecular machine for detecting exosomes according to claim 1, 3 or 4, wherein the ratio of the Substrate chain and the Motor chain on the GNP surface is 5:1 to 50: 1.
7. A DNA molecular machine for the detection of exosomes according to claim 1, 3 or 4, characterized in that the DNA molecular operating energy comes from the cleavage of specific sequences by the restriction enzyme Nt.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112763708A (en) * | 2020-12-24 | 2021-05-07 | 生物岛实验室 | Exosome detection method |
| CN113151584A (en) * | 2021-01-11 | 2021-07-23 | 南通大学 | SARS-CoV-2 detection kit and detection method |
| CN114395558A (en) * | 2022-01-17 | 2022-04-26 | 南通大学 | Magnetic bead-DNA probe, MC-LR detection biosensor, preparation method and application |
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2020
- 2020-07-09 CN CN202010655609.8A patent/CN111781180B/en active Active
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| HANYONG PENG ET.AL: "A microRNA-initiated DNAzyme motor operating in living cells", 《NATURE COMMUNICATIONS》 * |
| JUNBO CHEN ET.AL: "A Target-Triggered DNAzyme Motor Enabling Homogeneous,Amplified Detection of Proteins", 《ANALYTICAL CHEMISTRY》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112763708A (en) * | 2020-12-24 | 2021-05-07 | 生物岛实验室 | Exosome detection method |
| CN112763708B (en) * | 2020-12-24 | 2022-02-11 | 生物岛实验室 | Exosome detection method |
| CN113151584A (en) * | 2021-01-11 | 2021-07-23 | 南通大学 | SARS-CoV-2 detection kit and detection method |
| CN114395558A (en) * | 2022-01-17 | 2022-04-26 | 南通大学 | Magnetic bead-DNA probe, MC-LR detection biosensor, preparation method and application |
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