CN111521785A - Kit for quickly detecting cancer cell exosomes and preparation method and application thereof - Google Patents
Kit for quickly detecting cancer cell exosomes and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a kit for quickly quantifying cancer cell exosomes, which comprises a DNA tetrahedron-capture aptamer and a signal aptamer-poly G, wherein a DNA tetrahedron nanostructure is introduced to improve the application stability of the capture aptamer, and the nucleic acid type kit based on a double-aptamer 'sandwich' detection method is developed by combining a high-specificity aptamer with a high-sensitivity nuclease catalytic amplification signal technology. The kit provided by the invention realizes rapid and accurate quantification of cancer cell exosomes, realizes high-sensitivity and high-throughput detection of exosomes, and further realizes early diagnosis of cervical cancer.
Description
Technical Field
The invention belongs to the technical field of clinical diagnosis, and particularly relates to a nucleic acid type kit based on an enzyme-linked aptamer method.
Background
Exosomes (exosomes) derived from tumors are 30-100nm of small enveloped vesicles, play an important role in regulating and controlling the processes of tumorigenesis and tumor development, carry a large number of membrane proteins (tumor-specific molecules) derived from cancer cells on the surface, are enrichments of cancer biomarkers to a certain extent, have great in-vitro detection advantages, and can be used as biomarkers for early clinical diagnosis. Currently, the analysis of exosomes faces a number of problems: i) the exosome size is in the nanometer level, and the exosome is difficult to directly analyze by using cell analysis means such as a flow cytometer, and the like, while the fixing, marking and other means cause limited sensitivity and possibly damage the original structure; ii) exosome quantification is mostly limited to methods such as SPR (surface plasmon resonance) or electrochemistry, special instrument consumables are needed, and high-throughput screening of a large number of samples is difficult to meet.
At present, most of diagnostic kits for diseases such as hepatitis B are based on antigen-antibody reaction, enzyme-linked amplification reaction and 96 × enzyme label plate, and have the advantage of high-throughput screening. However, the preparation process of the antibody is complex and is easy to have batch-to-batch difference, which increases the difficulty of practical application.
Disclosure of Invention
The invention aims to provide a kit for quickly detecting cancer cell exosomes, a preparation method of the kit and specific application of the kit to make up for the defects of the prior art.
The aptamer (aptamer), also called aptamer and aptamer, has the advantages of high specificity, affinity and the like through a single-stranded oligonucleotide sequence obtained by screening through an in vitro SELEX technology, and can specifically recognize target proteins on the surface of an exosome, such as CD63 and the like, so that the aptamer can be combined with the exosome. Meanwhile, the aptamer can be synthesized without difference among batches, can receive different modification marks or is integrated into a nucleic acid nanostructure, achieves high-efficiency identification, and has wide application prospect.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention combines a high-specificity aptamer with a high-sensitivity nuclease combined amplification technology, introduces a DNA tetrahedral nano structure to improve the application stability of the captured aptamer, and develops a double-aptamer-based sandwich-type detection kit to realize high-sensitivity, high-specificity and high-throughput detection of cancer cell exosomes.
The method specifically comprises the following steps:
a kit for rapid quantification of cancer cell exosomes comprising DNA tetrahedron-capture aptamer and signal aptamer-poly G.
Further, the concentrations of the DNA tetrahedron-capture aptamer and the signal aptamer-poly G are both 2. mu.M.
Further, the kit also comprises Hemin (Hemin) and 3,3 ', 5, 5' -Tetramethylbenzidine (TMB).
The DNA tetrahedron-capture aptamer is characterized in that four single-stranded DNA sequences are designed, wherein three DNA sequences are modified by biotin (B), the other DNA sequence is modified by capture aptamer, and then a tetrahedron nano structure with B modification at the bottom and capture aptamer at the top is formed by molecular self-assembly.
The signal aptamer-poly G is obtained by connecting a signal aptamer DNA sequence with a poly G sequence.
The preparation method of the kit comprises the following steps:
(1) preparation of DNA tetrahedron-capture aptamers: firstly, four single-stranded DNA sequences are designed, wherein three DNA sequences have biotin (B) modification, the other DNA sequence has capture aptamer DNA sequence, and then a DNA tetrahedron structure with B modification at the bottom and capture aptamer at the top is formed through molecular self-assembly;
(2) on the basis that the bottom of a multi-well plate of the kit is coated with Streptavidin (SA), the DNA tetrahedral structure is fixed to the bottom of the multi-well plate through specific binding between SA-B;
(3) preparing a signal aptamer-poly G, wherein the signal aptamer DNA sequence is connected with a poly G sequence to obtain the signal aptamer-poly G.
The using and detecting method of the kit comprises the following steps:
(1) treating a sample to be detected, adding the treated sample into a porous plate of the kit, wherein the porous plate is fixed with a DNA tetrahedral structure, if an exosome exists, a capture aptamer is firstly combined with CD63 protein on the surface of the exosome, and removing supernatant;
(2) after washing the plate, adding a signal aptamer-poly G which can be combined with EpCAM protein on the surface of the exosome, forming a sandwich-type compound by the two aptamers and the exosome, and washing the plate;
(3) then, Hemin (Hemin) is added, a poly G sequence and the Hemin form a peroxide mimic enzyme which plays the activity of peroxidase (DNAzyme) and catalyzes a substrate to generate color change;
(4) and adding TMB, catalyzing the TMB by using an oxide mimic enzyme to generate color change from colorless to blue, changing the blue to yellow after the acid addition is stopped, and measuring the light absorption value at 450nm by using an enzyme-labeling instrument to realize the visual colorimetric detection of the exosome.
Further, the using and detecting method of the kit specifically comprises the following steps:
(1) construction and immobilization of capture aptamer-DNA tetrahedral nanostructure
First, 2. mu.M of the four tetrahedral strands (a, b, c, d) were added to the reaction system, and TM buffer (20mM Tris,100mM NaCl, and 5mM MgCl2, pH 8.0) was added to the system to provide buffer conditions required for DNA self-assembly, respectively. Then, performing molecular self-assembly by adopting a method of rapid heating and slow cooling, namely reacting for 5min at 95 ℃ to open the secondary structure of the DNA chain, and then slowly cooling to 25 ℃;
uniformly placing 1 mu g/mL of SA at the bottom of a multi-well plate, and washing the plate for later use after overnight; then adding 2 mu M of biotinylated DNA tetrahedron into a multi-hole plate containing SA respectively, reacting for 30min at room temperature, washing the plate for 3 times, removing unbound DNA tetrahedron, and successfully fixing the DNA tetrahedron containing the capture aptamer at the top on the multi-hole plate;
(2) colorimetric detection of exosomes
Will 106Adding each/mL of exosome into a multi-well plate containing a capture aptamer, reacting for 30min at 4 ℃, identifying CD63 protein on the surface of the exosome by the capture aptamer, and washing the plate for 3 times; then adding 0.25,0.5,1,2,4 μ M signal aptamer, reacting at 4 deg.C for 30min to identify EpCAM protein on the surface of exosome to form capture aptamer-exosome-signal aptamer sandwich structure, washing plate for 3 times(ii) a Then adding 40 mu M Hemin into the porous plate after the target reaction, reacting for 15min at room temperature to form DNAzyme, and washing the plate for 3 times;
then, 100. mu.L/well of TMB was added to the well plate, after reaction at room temperature for 15min, 50. mu.L of 2M sulfuric acid was added to terminate the reaction, and it was observed that the color of the solution changed from colorless to blue, and then changed to yellow after addition of acid, and then the light absorption value at 450nm was measured using a multifunctional microplate reader.
The sandwich type double-aptamer structure is characterized in that under the induction of a target (exosome), a capture aptamer and a signal aptamer are respectively subjected to specific recognition on membrane proteins on the surface of the exosome to form a capture aptamer-exosome-signal aptamer complex, wherein unbound signal aptamer in supernatant is removed through two-phase separation.
The kit can be used for detecting cancer cell exosomes.
Compared with the prior art, the invention has the beneficial effects that:
firstly, compared with single sample detection, the invention can realize high-throughput rapid screening and meet the screening requirements of a large number of samples. Secondly, the recognition factor, the signal factor and the like of the kit are all nucleic acids, so that the limitations of easy inactivation and the like of protein antibodies can be avoided. Finally, the introduction of a DNA tetrahedral structure is helpful for the immobilized aptamer to play a recognition role to the maximum extent; the introduction of a double aptamer sandwich structure and an enzyme-linked amplification technology is beneficial to improving the specificity and sensitivity of detection, and DNAzyme catalyzes TMB to generate color change, so that the response is quick, and visual detection can be realized. The invention can realize the rapid and accurate quantification of cancer cell exosomes.
Drawings
FIG. 1 is a schematic diagram of a diagnostic kit provided in the present invention;
FIG. 2 is a TEM representation result of the morphology of the Hela-exosomes extracted in the example of the present invention;
FIG. 3 is a graph showing the results of folding characterization of the DNA tetrahedron-capture aptamer complex structure according to the example of the present invention;
FIG. 4 is a graph of the results of optimizing tetrahedral-trapping aptamer concentration in an example of the invention;
FIG. 5 is a graph showing the results of optimizing the concentration of signal aptamer-poly G in an embodiment of the present invention;
FIG. 6 is a graph showing the results of the detection curve and the standard curve of exosomes in the example of the present invention.
Detailed Description
The invention will be further explained and illustrated by means of specific embodiments and with reference to the drawings.
Example 1
A nucleic acid type kit for rapid quantification of cancer cell exosomes based on an enzyme-linked aptamer method comprises two parts, namely construction of a sandwich type aptamer structure under target induction and optical signal transduction, and the principle is shown in figure 1, and the specific implementation steps are as follows:
(1) streptavidin (SA) is coated at the bottom of the ELISA plate (100. mu.L/well 1. mu.g/mL, overnight, after plate washing, no blocking is needed);
(2) the DNA tetrahedron assembled with the capture aptamer (100 muL/hole 2 muM, 30min, plate washing) is combined through SA-B to realize the directional fixation of the capture aptamer;
(3) then the capture aptamer can specifically recognize CD63 protein (100 mu L/hole concentration, 30min, plate washing) on the surface of the exosome, so as to capture the exosome;
(4) the signal aptamer is specifically combined with EpCAM protein on the surface of an exosome to form a capture aptamer-exosome-signal aptamer complex (100 mu L/well 2 mu M, 30min, plate washing);
(5) Poly-G was reacted with 40. mu.M Hemin at room temperature for 15min, poly G-tetrad exhibited peroxidase activity with the aid of Hemin, catalyzed TMB to produce a color reaction (from colorless to blue), reacted at 37 ℃ for 15min, and stopped by adding 50. mu.L of 2M sulfuric acid solution (from blue to yellow), giving the results shown in FIG. 1. Wherein, the higher the exosome concentration is, the deeper the yellow color is, and an enzyme-linked immunosorbent assay is further used for measuring an ultraviolet absorption value for accurate quantification.
(6) The tetrahedron-capture aptamer can be coated on the bottom of the porous plate in advance, and the two aptamers are specifically combined with the exosome to form a sandwich structure, wherein the process only needs about 1 hour; alternatively, the tetrahedral-trapping aptamers can be immobilized on the bottom of a multiwell plate at the time of use, and the subsequent formation of the "sandwich" structure under target induction requires only about 1.5 h.
Example 2
Taking cervical cancer, one of common female malignant tumors, as an example, a novel technology which is high in specificity and sensitivity and can carry out high-throughput screening on a large number of samples aiming at Hela cell exosomes is developed. The method specifically comprises the following steps:
(1) construction and immobilization of capture aptamer-DNA tetrahedral nanostructure
First, 4. mu.M of the four tetrahedral strands (a, b, c, d) were added to the reaction system, and TM buffer (20mM Tris,100mM NaCl, and 5mM MgCl) was added to the system2pH 8.0) provides the buffer conditions required for DNA self-assembly. Then, the molecular self-assembly is carried out by adopting a method of 'fast heating and slow cooling', namely, the reaction is carried out for 5min at the temperature of 95 ℃ so as to open the secondary structure of the DNA chain, and then the temperature is slowly reduced to 25 ℃.
The formation of tetrahedral nanostructures was verified using 3% agarose gel electrophoresis.
The construction of 2. mu.M DNA tetrahedrons and gel imaging results are shown in FIG. 3, with slower mobility as the number of DNA increases, indicating successful construction and high yield formation of tetrahedrons.
Uniformly placing 1 mu g/mL of SA at the bottom of a multi-well plate, and washing the plate for later use after overnight; then biotinylated DNA tetrahedron 0.25,0.5,1,2, 4. mu.M was added to the well plate containing SA, respectively, and reacted at room temperature for 30min, and then the plate was washed 3 times to remove unbound DNA tetrahedron, and the DNA tetrahedron with the capture aptamer on the top was successfully immobilized on the well plate.
(2) The results of colorimetric detection of exosomes and TEM characterization of the morphology of Hela-exosomes are shown in figure 2
Will 106Adding each/mL of exosome into a multi-well plate containing a capture aptamer, reacting for 30min at 4 ℃, identifying CD63 protein on the surface of the exosome by the capture aptamer, and washing the plate for 3 times; then adding 2 μ M signal aptamer, reacting at 4 deg.C for 30min, wherein the signal aptamer recognizes EpCAM protein on the surface of exosome to form capture aptamerLigand-exosome-signal aptamer 'sandwich' structure, plate washing 3 times; then adding 40 mu M Hemin into the multi-well plate after target reaction, reacting for 15min at room temperature to form DNAzyme, and washing the plate for 3 times.
Then, 100. mu.L/well of TMB was added to the well plate, and after reaction at room temperature for 15min, 50. mu.L of 2M sulfuric acid was added to terminate the reaction, and it was observed that the color of the solution changed from colorless to blue, and then changed to yellow after addition of acid, and then the light absorption value at 450nm was measured using a multifunctional microplate reader.
The absorbance change at 450nm, which is caused by the addition of capture aptamer-tetrahedra at different concentrations to the plate fixed under otherwise identical conditions, is shown in FIG. 4, and it is seen from the graph that the absorbance is caused to be the maximum at a fixed concentration of 2. mu.M, and thereafter a plateau is reached. Thus, the optimal immobilization concentration of the tetrahedral-trapping aptamer in the present invention is 2 μ M.
TABLE 1 sequence information of DNA tetrahedrons and aptamers used in the examples
Note:wave lineRepresents a nucleotide sequence of an aptamer; examples 2-6 all used the above sequence; examples 3-6 were further optimized for kit compositional concentrations and specific detection methods in this example, respectively.
Example 3
(1) Construction and immobilization of capture aptamer-DNA tetrahedral nanostructure
First, 2. mu.M of the four tetrahedral strands (a, b, c, d) were added to the reaction system, and TM buffer (20mM Tris,100mM NaCl, and 5mM MgCl) was added to the system2pH 8.0) provides the buffer conditions required for DNA self-assembly. Then, the molecular self-assembly is carried out by adopting a method of 'fast heating and slow cooling', namely, the reaction is carried out for 5min at the temperature of 95 ℃ so as to open the secondary structure of the DNA chain, and then the temperature is slowly reduced to 25 ℃.
Uniformly placing 1 mu g/mL of SA at the bottom of a multi-well plate for later use after overnight; then biotinylated DNA tetrahedrons 2. mu.M were added to the well plates containing SA, respectively, and reacted at room temperature for 30min, and then the plates were washed 3 times to remove unbound DNA tetrahedrons, and the DNA tetrahedrons containing the capture aptamers at the top were successfully immobilized on the well plates.
(2) Colorimetric detection of exosomes
Will 106Adding each/mL of exosome into a multi-well plate containing a capture aptamer, reacting for 30min at 4 ℃, identifying CD63 protein on the surface of the exosome by the capture aptamer, and washing the plate for 3 times; then adding 0.25,0.5,1,2 and 4 mu M of signal aptamer into the mixture respectively, reacting for 30min at 4 ℃, identifying EpCAM protein on the surface of the exosome by the signal aptamer to form a sandwich structure of capture aptamer-exosome-signal aptamer, and washing the plate for 3 times; then adding 40 mu M Hemin into the multi-well plate after target reaction, reacting for 15min at room temperature to form DNAzyme, and washing the plate for 3 times.
Then, 100. mu.L/well of TMB was added to the well plate, and after reaction at room temperature for 15min, 50. mu.L of 2M sulfuric acid was added to terminate the reaction, and it was observed that the color of the solution changed from colorless to blue, and then changed to yellow after addition of acid, and then the light absorption value at 450nm was measured using a multifunctional microplate reader.
FIG. 5 shows the absorbance change at 450nm under the same conditions, when different concentrations of signal aptamer-poly G specifically bind to EpCAM protein of exosome, and it is seen that the absorbance reaches a maximum at a signal aptamer-poly G concentration of 2. mu.M, and thereafter a plateau is reached. Therefore, the optimum concentration of the signal aptamer-poly G to be added in the present invention is 2. mu.M.
Example 4
(1) Construction and immobilization of capture aptamer-DNA tetrahedral nanostructure
First, 2. mu.M of the four tetrahedral strands (a, b, c, d) were added to the reaction system, and TM buffer (20mM Tris,100mM NaCl, and 5mM MgCl) was added to the system2pH 8.0) provides the buffer conditions required for DNA self-assembly. Then, the molecular self-assembly is carried out by adopting a method of 'fast heating and slow cooling', namely, the reaction is carried out for 5min at the temperature of 95 ℃ so as to open the secondary structure of the DNA chain, and then the temperature is slowly reduced to 25 ℃.
Uniformly placing 1 mu g/mL of SA at the bottom of a multi-well plate for later use after overnight; then biotinylated DNA tetrahedrons 2. mu.M were added to the well plates containing SA, respectively, and reacted at room temperature for 30min, and then the plates were washed 3 times to remove unbound DNA tetrahedrons, and the DNA tetrahedrons containing the capture aptamers at the top were successfully immobilized on the well plates.
(2) Colorimetric detection of exosomes
Respectively 10 are provided2,103,104,105,106,107,108,109,1010,1011Adding each/mL of exosome into a multi-well plate containing a capture aptamer, reacting for 30min at 4 ℃, identifying CD63 protein on the surface of the exosome by the capture aptamer, and washing the plate for 3 times; then respectively adding 2 mu M of signal aptamers into the mixture, reacting for 30min at 4 ℃, identifying EpCAM protein on the surface of an exosome by the signal aptamers to form a sandwich structure of capture aptamer-exosome-signal aptamer, and washing the plate for 3 times; then adding 40 mu M Hemin into the multi-well plate after target reaction, reacting for 15min at room temperature to form DNAzyme, and washing the plate for 3 times.
Then, 100. mu.L/well of TMB was added to the well plate, and after reaction at room temperature for 15min, 50. mu.L of 2M sulfuric acid was added to terminate the reaction, and it was observed that the color of the solution changed from colorless to blue, and then changed to yellow after addition of acid, and then the light absorption value at 450nm was measured using a multifunctional microplate reader.
The change of the absorbance at 450nm caused by the detection of exosomes of different concentrations by using the nucleic acid-type kit is shown in FIG. 6, and as can be seen from FIG. 6, the absorbance gradually increases as the exosome concentration increases; and is at 103～109In the range of per mL, the logarithm of the exosome concentration and the absorbance value form a certain linear relation, and the linear formula is as follows: y 0.0735x-0.0390, R20.9869. The kit can be used for detecting exosome.
Example 5
(1) Construction and immobilization of capture aptamer-DNA tetrahedral nanostructure
First, 2. mu.M of the four tetrahedral strands (a, b, c, d) were added to the reaction system, and TM buffer (20mM Tris, 100. mu.M) was added to the systemmM NaCl,and 5mM MgCl2pH 8.0) provides the buffer conditions required for DNA self-assembly. Then, the molecular self-assembly is carried out by adopting a method of 'fast heating and slow cooling', namely, the reaction is carried out for 5min at the temperature of 95 ℃ so as to open the secondary structure of the DNA chain, and then the temperature is slowly reduced to 25 ℃.
Uniformly placing 1 mu g/mL of SA at the bottom of a multi-well plate for later use after overnight; then biotinylated DNA tetrahedrons 2. mu.M were added to the well plates containing SA, respectively, and reacted at room temperature for 30min, and then the plates were washed 3 times to remove unbound DNA tetrahedrons, and the DNA tetrahedrons containing the capture aptamers at the top were successfully immobilized on the well plates.
(2) Colorimetric detection of exosomes
Adding a buffer system without exosome into a porous plate containing a capture aptamer, reacting for 30min at 4 ℃, and washing the plate for 3 times; then adding 2 mu M signal aptamers into the mixture respectively, reacting for 30min at 4 ℃, and washing the plate for 3 times by signals; then, 40. mu.M Hemin was added to the reacted multi-well plate, and reacted at room temperature for 15min to form DNAzyme, and the plate was washed 3 times.
Then, 100. mu.L/well of TMB was added to the well plate, and after reaction at room temperature for 15min, the reaction was terminated by adding 50. mu.L of 2M sulfuric acid, and it was observed that the color of the solution remained almost colorless, and then the light absorption value at 450nm was measured using a multi-functional microplate reader. And averaging the light absorption values measured in parallel for 6 times, and substituting the light absorption values into a linear formula: y is 0.0735x-0.0390, and the detection limit of the kit is calculated to be 1019/mL, which indicates that the kit has higher detection sensitivity.
Example 6
(1) Construction and immobilization of capture aptamer-DNA tetrahedral nanostructure
First, 2. mu.M of the four tetrahedral strands (a, b, c, d) were added to the reaction system, and TM buffer (20mM Tris,100mM NaCl, and 5mM MgCl) was added to the system2pH 8.0) provides the buffer conditions required for DNA self-assembly. Then, the molecular self-assembly is carried out by adopting a method of 'fast heating and slow cooling', namely, the reaction is carried out for 5min at the temperature of 95 ℃ so as to open the secondary structure of the DNA chain, and then the temperature is slowly reduced to 25 ℃.
Uniformly placing 1 mu g/mL of SA at the bottom of a multi-well plate for later use after overnight; then biotinylated DNA tetrahedrons 2. mu.M were added to the well plates containing SA, respectively, and reacted at room temperature for 30min, and then the plates were washed 3 times to remove unbound DNA tetrahedrons, and the DNA tetrahedrons containing the capture aptamers at the top were successfully immobilized on the well plates.
(2) Colorimetric detection of exosomes
Taking 9 samples, respectively containing 104,106,108，104,106,108，104,106,108Adding the reaction liquid of each/mL of exosomes into a porous plate containing a capture aptamer, reacting for 30min at 4 ℃, identifying the CD63 protein on the surface of the exosomes by the capture aptamer, and washing the plate for 3 times; then respectively adding 2 mu M of signal aptamers into the mixture, reacting for 30min at 4 ℃, identifying EpCAM protein on the surface of an exosome by the signal aptamers to form a sandwich structure of capture aptamer-exosome-signal aptamer, and washing the plate for 3 times; then adding 40 mu M Hemin into the multi-well plate after target reaction, reacting for 15min at room temperature to form DNAzyme, and washing the plate for 3 times.
Then, 100. mu.L/well of TMB was added to the well plate, and after reaction at room temperature for 15min, 50. mu.L of 2M sulfuric acid was added to terminate the reaction, and it was observed that the color of the solution changed from colorless to blue, and then changed to yellow after addition of acid, and then the light absorption value at 450nm was measured using a multifunctional microplate reader.
Table 2 shows the change of absorbance at 450nm caused by using the nucleic acid type kit to detect 9 samples containing exosomes with different concentrations, and it can be seen from the table that the absorbance gradually increases with the increase of the exosome concentration; and substituting the measured light absorption value into a linear formula, and then obtaining the standard addition recovery rate of the kit for detecting the exosome, wherein the recovery rate is found to be between 84.6% and 116%, which indicates that the accuracy of the kit for detecting the exosome is higher.
TABLE 2 recovery rate of exosomes detected by nucleic acid type kit
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (9)
1. A kit for rapid quantification of cancer cell exosomes, comprising a DNA tetrahedron-capture aptamer and a signal aptamer-poly G.
2. The kit of claim 1, wherein the DNA tetrahedron-capture aptamer and signal aptamer-poly G are each present at a concentration of 2 μ Μ.
3. The kit of claim 1, wherein the DNA tetrahedron-capture aptamer is designed into four single-stranded DNA sequences, wherein three DNA sequences carry biotin modification, and the other DNA sequence carries capture aptamer DNA sequence, and then the tetrahedral nanostructure with biotin modification at the bottom and capture aptamer at the top is formed by molecular self-assembly.
4. The kit of claim 1, wherein the signal aptamer-poly G is obtained by ligating a signal aptamer DNA sequence to a poly G sequence.
5. The method for preparing the kit according to claim 1, comprising:
(1) preparation of DNA tetrahedron-capture aptamers: firstly, four single-stranded DNA sequences are designed, wherein three DNA sequences have biotin modification, the other DNA sequence has capture aptamer DNA sequence, and then a DNA tetrahedron structure with biotin modification at the bottom and capture aptamer at the top is formed through molecular self-assembly;
(2) on the basis that the bottom of a porous plate of the kit is coated by streptavidin, the DNA tetrahedral structure is fixed to the bottom of the porous plate through specific combination between SA-B;
(3) preparing a signal aptamer-poly G, wherein the signal aptamer DNA sequence is connected with a poly G sequence to obtain the signal aptamer-poly G.
6. The method for detecting a kit according to claim 1, which comprises:
(1) treating a sample to be detected, adding the treated sample into a porous plate of the kit, wherein the porous plate is fixed with a DNA tetrahedral structure, if an exosome exists, a capture aptamer is firstly combined with CD63 protein on the surface of the exosome, and removing supernatant;
(2) after washing the plate, adding a signal aptamer-poly G which can be combined with EpCAM protein on the surface of the exosome, forming a sandwich-type compound by the two aptamers and the exosome, and washing the plate;
(3) then, Hemin, poly G sequence and Hemin are added to form a peroxide mimic enzyme which exerts peroxidase activity and catalyzes a substrate to generate color change;
(4) and adding TMB, catalyzing the TMB by using an oxide mimic enzyme to generate color change from colorless to blue, changing the blue to yellow after the acid addition is stopped, and measuring the light absorption value at 450nm by using an enzyme-labeling instrument to realize the visual colorimetric detection of the exosome.
7. The detection method according to claim 6, wherein the "sandwich" complex is characterized in that the capture aptamer and the signal aptamer are specifically recognized in membrane proteins on the surface of the exosome respectively under the induction of the target exosome to form a capture aptamer-exosome-signal aptamer complex, and unbound signal aptamer in supernatant is removed by two-phase separation.
8. The detection method according to claim 6, specifically comprising:
(1) construction and immobilization of capture aptamer-DNA tetrahedral nanostructure
First, 2. mu.M of tetrahedral four-strands were added to the reaction system, respectively, and TM buffer, 20mM Tris,100mM NaCl, and 5mM MgCl were added to the system2pH 8.0, providing buffer conditions for DNA self-assembly;
then, performing molecular self-assembly by adopting a method of 'rapid heating-slow cooling', namely reacting at 95 ℃ for 5min to open the secondary structure of the DNA chain, and then slowly cooling to 25 ℃;
uniformly placing 1 mu g/mL of SA at the bottom of a multi-well plate, and washing the plate for later use after overnight; then adding 2 mu M of biotinylated DNA tetrahedron into a multi-hole plate containing SA respectively, reacting for 30min at room temperature, washing the plate for 3 times, removing unbound DNA tetrahedron, and successfully fixing the DNA tetrahedron containing the capture aptamer at the top on the multi-hole plate;
(2) colorimetric detection of exosomes
Will 10^ a6Adding each/mL of exosome into a multi-well plate containing a capture aptamer, reacting for 30min at 4 ℃, identifying CD63 protein on the surface of the exosome by the capture aptamer, and washing the plate for 3 times; then adding 0.25,0.5,1,2 and 4 mu M of signal aptamer into the mixture respectively, reacting for 30min at 4 ℃, identifying EpCAM protein on the surface of the exosome by the signal aptamer to form a sandwich structure of capture aptamer-exosome-signal aptamer, and washing the plate for 3 times; then adding 40 mu M Hemin into the porous plate after target reaction, and reacting for 15min at room temperature to formDNAzyme, wash plate 3 times;
then, 100. mu.L/well of TMB was added to the well plate, and after reaction at room temperature for 15min, 50. mu.L of 2M sulfuric acid was added to terminate the reaction, and it was observed that the color of the solution changed from colorless to blue, and then changed to yellow after addition of acid, and then the light absorption value at 450nm was measured using a multifunctional microplate reader.
9. The kit of claim 1 can be used for the detection of cancer cell exosomes.
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| US20190048347A1 (en) * | 2016-11-02 | 2019-02-14 | Sichuan University | Nucleic acid aptamer as1411 modified dna tetrahedron and preparation method thereof |
| CN110305770A (en) * | 2019-07-17 | 2019-10-08 | 中国科学院上海高等研究院 | A kind of micro-fluidic chip of DNA nanostructure modification is sensed and its prepared for optical bio and application |
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| CN102827836A (en) * | 2012-06-11 | 2012-12-19 | 中国科学院成都生物研究所 | Oligonucleotide probe, and method for detecting target molecule through using it |
| US20190048347A1 (en) * | 2016-11-02 | 2019-02-14 | Sichuan University | Nucleic acid aptamer as1411 modified dna tetrahedron and preparation method thereof |
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