CN114634973A - Tumor exosome detection method based on aptamer recognition - Google Patents
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Abstract
A tumor exosome detection method based on nucleic acid aptamer recognition utilizes a hairpin-shaped probe H1, a first signal probe H2 modified by fluorescent molecule FAM and a second signal probe H3 modified by TAMRA, tumor exosomes are combined with a nucleic acid aptamer part of the hairpin-shaped probe H1, the hairpin-shaped probe H1 is opened, a trigger sequence I required by hybridization chain reaction is released, the first signal probe H2 and the second signal probe H3 are triggered to perform hybridization chain reaction to form a long double chain, the fluorescent molecule FAM of the first signal probe H2 and the fluorescent molecule TAMRA on the second signal probe H3 are close to each other on the formed double chain, fluorescence resonance energy transfer occurs, and quantitative detection can be carried out on the tumor exosomes through the ratio of the fluorescent molecules FAM to the TAMRA. The detection specificity is good, and the applicability in complex samples is improved. And the sensitivity is higher, the complicated separation process required by most of the existing exosome detections and errors possibly caused by the separation process are avoided, and the method has remarkable social and economic benefits.
Description
Technical Field
The utility model belongs to the technical field of exosome detection, and particularly relates to a tumor exosome detection method based on aptamer recognition.
Background
Lung cancer is a common malignant tumor, and the morbidity and mortality of lung cancer are both in the prostate of malignant tumor. A liquid biopsy technique for detecting biomarkers secreted from tumor tissues into body fluids such as blood is a new method for tumor diagnosis developed in recent years. The method has the advantages of small sampling wound, convenient detection and the like, and is an ideal method for early diagnosis of tumors. In tumor fluid biopsies, the selection of the detection marker is crucial. The exosome is a lipid bilayer structure vesicle with the diameter of 30-150 nm and actively secreted by cells, is an important communication medium for mediating the transmission of proteins, nucleic acids and metabolites among cells, and researches show that the exosome is closely related to the processes of the occurrence, the metastasis and the like of lung cancer. The tumor exosomes carry information of a large number of tumor cells, for example, the surfaces of the tumor exosomes contain transmembrane proteins and receptors which are specific to a plurality of tumor cells, and molecules such as a large number of tumor miRNAs, DNAs (deoxyribonucleic acids), proteins and the like are loaded in the tumor exosomes, so the tumor exosomes in blood are tumor markers with great application potential.
The current exosome detection methods mainly include Nanoparticle Tracking Analysis (NTA), enzyme-linked immunosorbent assay (ELISA) immunoblotting (Western blot, WB), and the like, but most of these methods have low sensitivity, complicated operation, large required sample size, and are difficult to meet the requirements of clinical detection. In order to overcome the defects of the detection method, various high-sensitivity detection methods are developed, such as Surface Plasma Resonance (SPR), dark-field microscopic imaging, surface enhanced Raman scattering and the like, however, most of the methods need special equipment, and certain limitations exist in the aspect of application and popularization.
Disclosure of Invention
In view of the above situation, in order to overcome the defects of the prior art, the present invention aims to provide a method for detecting tumor exosomes by nucleic acid aptamer recognition, which can effectively solve the problem of detection result error caused by poor specificity and low sensitivity in the aspect of detecting tumor exosomes in the prior art.
In order to achieve the above objects, the present invention provides a method for detecting tumor exosomes identified by aptamer, which comprises using hairpin probe H1 for identifying tumor exosomes, first signal probe H2 modified by fluorescent molecule FAM and second signal probe H3 modified by TAMRA, when tumor exosomes are present, binding the tumor exosomes to the aptamer portion of hairpin probe H1, opening hairpin probe H1, releasing trigger sequence I required for hybridization chain reaction, triggering the first signal probe H2 and the second signal probe H3 to perform hybridization chain reaction to form a long double chain, bringing the fluorescent molecule FAM of the first signal probe H2 and the fluorescent molecule TAMRA of the second signal probe H3 close to each other on the formed double chain, and generating fluorescence resonance energy transfer, with the increase of fluorescent signal of the fluorescent molecule TAMRA, and the fluorescent signal of the fluorescent molecule FAM decreasing, the fluorescence difference between the two is positively correlated with the concentration of tumor exosomes in the system, and the tumor exosomes can be quantitatively detected through the fluorescence signal ratio of fluorescent molecules FAM and TAMRA.
Further, the hairpin probe H1 is formed by the aptamer sequence apt and the trigger sequence I of hybridization chain reaction through extending the sequence to form a hairpin structure, and the trigger sequence I is in a locked state due to intramolecular nucleic acid hybridization; after the aptamer sequence apt in the hairpin probe H1 is bound to tumor exosomes, the trigger sequence I required for hybridization chain reaction is released.
The sequences of the first signal probe H2 and the second signal probe H3 are partially complementary, and the 3' end of the first signal probe H2 and the ring part of the second signal probe H3 are respectively modified with fluorescent molecules FAM and TAMRA which can generate fluorescence resonance energy transfer.
A tumor exosome detection method based on aptamer recognition specifically comprises the following steps:
(1) preparing a recognition probe: dissolving a probe containing the aptamer sequence apt and the trigger sequence I in 5 mM MgCl containing 250 nM glucose2·6H2In PBS buffer solution of O, the concentration of the probe is 400 nM, the solution is placed on a vortex mixer for 30 s and mixed evenly, then placed in a water bath, gradually heated to 90 ℃, taken out after 10 min, slowly cooled to room temperature, and a hairpin-shaped probe H1, namely a recognition probe, is obtained by utilizing complementary hybridization reaction of nucleic acid;
(2) and preparing a signal probe: with 5 mM MgCl containing 250 nM glucose2·6H2Dissolving a first signal probe H2 modified by fluorescent molecule FAM and a second signal probe H3 modified by fluorescent molecule TAMRA in PBS buffer solution of O respectively, wherein the concentrations of the first signal probe H2 and the second signal probe H3 are both 800 nM, uniformly mixing the two in a vortex mixer for 30 s, then placing the mixture in a water bath, gradually raising the temperature to 90 ℃, taking out the mixture after 10 min, slowly cooling the mixture to room temperature, then respectively mixing the mixture with the same amount of the first signal probe H2 and the second signal probe H3, and uniformly mixing the mixture in the vortex mixer for 30 s to obtain signal probes;
(3) tumor exosome detection: adding the recognition probe and the signal probe prepared in the step into a cell solution, wherein the ratio of the concentration of the recognition probe to the concentration of the signal probe is 1:4, reacting for 3h, when no tumor exosome exists, the trigger sequence I in the recognition probe cannot trigger the hybridization chain reaction of the signal probe, at the moment, the FAM and the TAMRA are far away, the FRET signal between the FAM and the TAMRA cannot be detected, and when the tumor exosome exists, detecting the fluorescence emission intensity of the FAM and the TAMRA by using a fluorescence spectrophotometer under the excitation of 480 nm excitation light, so as to realize the quantitative detection of the tumor exosome.
The tumor exosome is lung cancer A549 cells or other cell exosomes capable of being combined with the aptamer.
The sequence of the aptamer sequence apt is: 5'-TTT ATG GGT GGG TGG GGG GTT TTT-3', respectively;
the sequence of trigger sequence I is: 5'-CACCCATAAAGACTGATGTTGA-3', respectively;
the sequence of the first signal probe H2 for modifying the fluorescent molecule FAM is as follows:
5’- TCA ACA TCA GTC TTT ATG GGT GCA AGC ACC CAT AAA GAC TGA -3’-FAM；
the sequence of the second signal probe H3 for modifying the fluorescent molecule TAMRA is as follows:
5’- C ACC CAT AAA GAC TGA TGT TGA T（TAMRA）CA GTC TTT ATG GGT GCT TG -3’。
the method is simple, easy to operate and good in detection specificity, can reduce false positive signals to a great extent, and improves the applicability in complex samples. And the sensitivity is higher, the nucleic acid hybridization chain reaction can be continuously extended once triggered, a large number of double chains which are alternately hybridized are generated, so that FRET signals are multiplied, the purpose of signal amplification is achieved, the complicated separation process required by most of the existing exosome detection and errors possibly caused by the separation process are avoided, and the social and economic benefits are remarkable.
Drawings
FIG. 1 is a schematic diagram of tumor exosome detection according to the present invention.
FIG. 2 is a line graph showing the results of the fluorescence resonance energy transfer and the A549 cell-derived exosomes at different concentrations according to the present invention.
FIG. 3 is a bar graph of the detection of exosomes from different cell sources of the present invention.
Detailed Description
The following detailed description of the embodiments of the utility model is provided in connection with the accompanying drawings and the detailed description.
The utility model relates to a tumor exosome detection method identified by a nucleic acid aptamer, which comprises the following steps:
(1) lung cancer A549 cell culture and exosome extraction: using an RPMI 1640 culture medium containing 10% by mass of exosome-free fetal bovine serum FBS as a cell culture solution, supplementing penicillin or streptomycin with the mass concentration of 1% into the culture solution, culturing lung cancer A549 cells in a carbon dioxide incubator with the volume concentration of 5%, wherein the temperature is 37 ℃, and collecting cell supernatant when the cells grow to 80-90%; centrifuging the collected cell supernatant by a centrifugal ultrafiltration method to purify the cell exosomes, namely centrifuging the collected cell supernatant for 30min under the centrifugation condition of 2500 g, filtering the supernatant by an ultrafiltration membrane of 0.22 m to remove cell debris, centrifuging the collected liquid by an ultrafiltration tube of 100 kDa for 30min under the condition of 5000 g, dissolving the collected solution containing the exosomes in PBS of pH 7.4, and storing the solution at-80 ℃ for later use;
(2) preparing a recognition probe: dissolving a probe containing the aptamer sequence apt and the trigger sequence I in a solution containing 250 nM glucose and 5 mM MgCl2·6H2In PBS buffer solution of O, the concentration of the probe is 400 nM, the solution is put on a vortex mixer for 30 s and mixed evenly, then the solution is put in a water bath, the temperature is gradually raised to 90 ℃, the solution is taken out after 10 min, the solution is slowly cooled to the room temperature, and a hairpin-shaped probe H1, namely a recognition probe, is obtained by utilizing the complementary hybridization reaction of nucleic acid;
(3) and preparing a signal probe: with 5 mM MgCl containing 250 nM glucose2·6H2Dissolving a first signal probe H2 modified by fluorescent molecule FAM and a second signal probe H3 modified by fluorescent molecule TAMRA in PBS buffer solution of O respectively, wherein the concentrations of the first signal probe H2 and the second signal probe H3 are both 800 nM, uniformly mixing the two in a vortex mixer for 30 s, then placing the mixture in a water bath, gradually raising the temperature to 90 ℃, taking out the mixture after 10 min, slowly cooling the mixture to room temperature, then respectively mixing the mixture with the same amount of the first signal probe H2 and the second signal probe H3, and uniformly mixing the mixture in the vortex mixer for 30 s to obtain signal probes;
(4) drawing a standard curve: respectively adding the prepared recognition probe and the prepared signal probe into exosomes derived from lung cancer A549 cells with different concentrations, wherein the concentration of the recognition probe and the concentration of the signal probe are respectively 50nM and 200nM, reacting for 3h, detecting the fluorescence emission intensity of FAM at 522 nM and TAMRA at 580nM under the excitation of 480 nM exciting light by using a fluorescence spectrophotometer, taking the concentration of the exosomes as a horizontal ordinate, taking the ratio of the fluorescence intensity at 522 nM to that at 580nM as a vertical ordinate, and drawing a standard curve, wherein R is the ratio of the fluorescence intensity at the 522 nM to that at the 580nM, and the ratio of the fluorescence intensity of the exosomes is the same as the standard curve2Is 0.992;it can be concluded that the greater the concentration of lung cancer A549 cells, the greater the fluorescence intensity ratio;
(5) selective response study:
culturing and collecting a normal cell culture solution supernatant, respectively collecting and extracting exosomes by adopting the same method as the step (1), taking the obtained exosomes of different cell sources as a control, enabling the total amount of the exosomes contained in each solution to be detected to be equivalent, adding a detection probe with the same concentration under the same condition, recording the fluorescence intensity values of each sample at the emission wavelengths of fluorescent molecules FAM and TAMRA by using a fluorescence instrument, and taking the ratio of the fluorescence intensity values to the TAMRA as a signal of the fluorescence resonance energy transfer degree of a system to obtain that the fluorescence resonance energy transfer signals generated by the exosomes of the control cell sources are weaker and far lower than the signal intensity generated by the exosomes of the lung cancer A549 cell sources with the same concentration; the kit shows that the kit can specifically identify and detect the exosomes from the lung cancer A549 cells, does not respond to the exosomes from other different cell sources, and detects the high selectivity of the lung cancer A549 cell exosomes;
(6) and detecting exosomes in the complex biological sample: and (2) detecting fluorescence resonance energy signals of the system in PBS and 10% FBS respectively by taking the Bst cell exosomes as an interferent to obtain that the fluorescence signals of an exosome group containing a Bst cell source and a group without the interferent exosomes have no obvious difference, and the results in 10% FBS and the results in PBS both show interference, which indicates that the detection of the target tumor cell exosomes has no interference.
The utility model, when embodied, is given by the following examples.
Firstly synthesizing a hairpin probe H1, a first signal probe H2 and a second signal probe H3, wherein the hairpin probe H1 consists of an aptamer sequence and a trigger sequence I of hybridization chain reaction, and locks the sequence by forming a hairpin structure through sequence design, H2 and H3 are extension sequences of the hybridization chain reaction,
aptamer sequence apt:
5'-TTT ATG GGT GGG TGG GGG GTT TTT-3', as shown in sequence listing 1;
trigger sequence I:
5'-CACCCATAAAGACTGATGTTGA-3', as shown in sequence listing 2;
hairpin probe H1:
5'-CAC CCA TAA AGA CTG ATG TTG ATC AGT CTT TAT GGG TGG G GGG GGG TTT TTT T-3', as shown in sequence listing 3;
first signal probe H2:
5'-TCA ACA TCA GTC TTT ATG GGT GCA AGC ACC CAT AAA GAC TGA-3', as shown in sequence listing 4;
second signaling probe H3:
5 '-C ACC CAT AAA GAC TGA TGT TGA T (F2) CA GTC TTT ATG GGT GCT TG-3' as shown in 5 in the sequence table;
the 3' end of the first signaling probe H2 and the loop part of the second signaling probe H3 are respectively modified with a fluorescent group FAM (F1) and a TAMRA (F2) which can generate fluorescence resonance energy transfer, as shown in FIG. 1, that is:
the sequence of the first signal probe H2 for modifying the fluorescent molecule FAM is as follows:
5’- TCA ACA TCA GTC TTT ATG GGT GCA AGC ACC CAT AAA GAC TGA -3’-FAM；
the sequence of the second signal probe H3 for modifying the fluorescent molecule TAMRA is as follows:
5’- C ACC CAT AAA GAC TGA TGT TGA T（TAMRA）CA GTC TTT ATG GGT GCT TG -3’
the aptamer for specifically recognizing the exosome from the lung cancer A549 cell is used as a recognition molecule, the recognition binding effect of the aptamer and the exosome from the tumor cell is combined with the characteristic that the hybridization chain reaction of nucleic acid needs to be triggered by a specific sequence, the characteristic that a double chain is formed by intermolecular hybridization after the hybridization chain reaction occurs is utilized, and the fluorescence of Fluorescence Resonance Energy Transfer (FRET) between two types of fluorescent groups modified on a hairpin chain of the hybridization chain reaction is used as a signal reading mode, so that the high-sensitivity detection method of the exosome from the lung cancer A549 cell is developed.
When no lung cancer A549 cell-derived exosome is present, the hybridization chain trigger sequence I in the hairpin probe H1 cannot trigger the hybridization chain reaction between the first signaling probe H2 and the second signaling probe H3, and when F1 on the first signaling probe H2 and F2 on H3 are far away, the FRET signal between F1 and F2 cannot be detected. When the lung cancer cell exosome exists, the lung cancer cell exosome is combined with the aptamer part of the hairpin-shaped probe H1, the hairpin is opened, the trigger sequence I required by the hybridization chain reaction is released, the hybridization chain reaction of the first signal probe H2 and the second signal probe H3 is initiated to form a long double chain, at the moment, the F1 and the F2 on the first signal probe H2 and the second signal probe H3 are close to each other and generate FRET on the formed double chain, and the fluorescence signal of the F2 is increased, and as the hybridization chain reaction can generate a large number of double chains formed by alternately hybridizing the first signal probe H2 and the second signal probe H3, a large number of close F1 and F2 can be generated on the formed double chain, so that the FRET signal can be doubled, and the purpose of signal amplification is achieved. In specific tests, the utility model obtains very good effect, and the specific test data are as follows:
1. cell culture and exosome extraction:
firstly, using an RPMI 1640 culture medium containing 10% by mass of exosome-free fetal bovine serum FBS as a cell culture solution, supplementing 1% by mass of penicillin or streptomycin into the culture solution, culturing lung cancer A549 cells in a carbon dioxide incubator containing 5% by volume of the cells at 37 ℃, and collecting cell supernatant when the cells grow to 80-90%; . The above processes are all operated under aseptic conditions.
And separating and purifying the extracellular exosomes in the collected cell supernatant by using a centrifugal ultrafiltration method reported in the literature. The method comprises the following specific steps: the collected cell supernatant was centrifuged at 2500 g for 30min, then the supernatant was filtered through a 0.22 m ultrafiltration membrane to remove cell debris, then the collected solution was centrifuged at 5000 g for 30min through a 100 kDa ultrafiltration tube, and the collected exosome-containing solution was dissolved in PBS at pH 7.4 and stored at-80 ℃ for further use.
2. Preparation of recognition probes:
the DNA nucleic acid sequences used in the experiments were synthesized by Shanghai Biotechnology engineering Co., Ltd, and the probes containing the aptamer sequence apt and the trigger sequence I were dissolved in 5 mM MgCl and 250 nM glucose2·6H2And (2) in PBS (phosphate buffer solution) of O, wherein the concentration of the probe is 400 nM, the solution is placed on a vortex mixer for 30 s and mixed uniformly, then the mixture is placed in a water bath, the temperature is gradually increased to 90 ℃, the mixture is taken out after 10 min, the mixture is slowly cooled to room temperature, and a hairpin-shaped probe H1, namely a recognition probe, is formed by utilizing complementary hybridization reaction of nucleic acid.
3. Drawing a standard curve:
with 5 mM MgCl containing 250 nM glucose2·6H2And (3) respectively dissolving a first signal probe H2 modified by fluorescent molecule FAM and a second signal probe H3 modified by fluorescent molecule TAMRA in PBS buffer solution of O, wherein the concentrations of the first signal probe H2 and the second signal probe H3 are both 800 nM, uniformly mixing the two in a vortex mixer for 30 s, then placing the mixture in a water bath, gradually raising the temperature to 90 ℃, taking out the mixture after 10 min, slowly cooling the mixture to room temperature, then respectively mixing the mixture with the same amount of the first signal probe H2 and the second signal probe H3, and uniformly mixing the mixture in the vortex mixer for 30 s to obtain the signal probes.
4. The method comprises the following steps:
adding exosomes derived from A549 cells with different concentrations, adding the recognition probe H1 and the signal probes H2 and H3 prepared in the above steps, wherein the concentration of the recognition probe and the concentration of the signal probe are respectively 50nM and 200nM, after reacting for 3H, detecting the fluorescence emission intensity of FAM at 522 nM and TAMRA at 580nM under excitation of excitation light at 480 nM in a reaction solution by using a fluorescence spectrophotometer, drawing a standard curve by taking the concentration of the exosomes as a horizontal coordinate and the ratio of the fluorescence intensity at 522 nM and 580nM as a vertical coordinate, and drawing R, wherein R is a standard curve20.992, as shown in FIG. 2. It can be seen that the fluorescence intensity ratio is larger as the concentration of lung cancer a549 cells is larger.
5. Selective response investigation:
culturing and collecting culture solution supernatants of normal human embryonic lung fibroblast MRC-5, normal mammary gland cell Hs578Bst and liver cancer cell HepG2, collecting and extracting exosomes therein by adopting the same method as the step 1, taking the obtained exosomes of different cell sources as a reference, adjusting the volume of the added sample to ensure that the total amount of the exosomes contained in each solution to be detected is equivalent, adding detection probes with the same concentration under the same condition, recording the fluorescence intensity value of each sample at the emission wavelength of F1 and F2 by a fluorescence instrument, and taking the ratio of the two values as a signal of the fluorescence resonance energy transfer degree of the system. The results are shown in fig. 3, and the fluorescence resonance energy transfer signals generated by the exosomes derived from the control cells are all weak and are far lower than the signal intensity generated by the exosomes derived from the a549 cells at the same concentration. The results show that the detection system can specifically identify and detect the exosomes derived from the A549 cells, but does not respond to the exosomes derived from other different cells, and the high selectivity of the strategy for detecting the tumor exosomes is embodied.
6. Detection of exosomes in complex biological samples:
the fluorescence resonance energy signals of the system are detected in PBS and 10% FBS respectively by taking Bst cell exosomes as interferents, and the fluorescence signals of the exosome group containing Bst cell sources and the group without interfering exosomes have no obvious difference, which indicates that the detection of the target tumor cell exosomes is not interfered, and the result in 10% FBS is similar to that in PBS, and no obvious interference occurs.
In summary, the utility model is based on the characteristics that the aptamer recognizes and binds to tumor exosomes and the hybridization chain reaction of nucleic acid needs a specific sequence to trigger, the binding of the aptamer and the tumor exosomes is designed as the triggering condition of the nucleic acid hybridization chain reaction, and the property that the distance between a fluorescence donor molecule and a fluorescence acceptor molecule is reduced after the hybridization chain reaction occurs to generate fluorescence resonance energy transfer is combined, so that the utility model is an innovation in the detection of the high-sensitivity and high-specificity lung cancer exosomes.
The method is simple, easy to operate and good in detection specificity, and only after the aptamer is specifically combined with the tumor exosome, the trigger sequence of the nucleic acid hybridization chain reaction is released, so that the nucleic acid hybridization chain reaction is caused, the distance between fluorescent groups on two nucleic acid molecules is shortened due to hybridization aggregation, a FRET fluorescent signal is generated, the false positive signal is reduced to a great extent, and the applicability of the method in a complex sample is improved. Moreover, the sensitivity is high, and the nucleic acid hybridization chain reaction can be continuously extended once triggered to generate a large number of double chains which are alternately hybridized, so that the FRET signal is multiplied, and the purpose of signal amplification is achieved. In addition, the utility model belongs to homogeneous detection, does not need a complicated separation process, adopts sequence design and a signal generation mode initiated by tumor exosomes, and can avoid the complicated separation process required by most of the prior exosome detection and errors possibly caused by the complicated separation process. The utility model also shows better detection stability in a serum sample, provides a new idea for the development of tumor liquid biopsy, and has remarkable social and economic benefits.
Sequence listing
<110> Zhengzhou university
<120> a tumor exosome detection method based on aptamer recognition
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<170> SIPOSequenceListing 1.0
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cacccataaa gactgatgtt gatcagtctt tatgggtggg ggggggtttt ttt 53
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cacccataaa gactgatgtt gatcagtctt tatgggtgct tg 42
Claims (7)
1. A tumor exosome detection method of nucleic acid aptamer recognition is characterized in that a hairpin probe H1 for recognizing tumor exosomes, a fluorescent molecule FAM modified first signal probe H2 and a TAMRA modified second signal probe H3 are utilized, when the tumor exosomes exist, the tumor exosomes are combined with the aptamer part of the hairpin probe H1, the hairpin probe H1 is opened, a trigger sequence I required by hybridization chain reaction is released, the first signal probe H2 and the second signal probe H3 are triggered to perform hybridization chain reaction to form a long double chain, the fluorescent molecule FAM of the first signal probe H2 and the fluorescent molecule TAMRA on the second signal probe H3 are close to each other on the formed double chain, fluorescence resonance energy transfer occurs, the fluorescence signal of the fluorescent molecule FAM is reduced along with the increase of the fluorescent molecule TAMRA, the fluorescence signal of the fluorescent molecule FAM is positively correlated with the concentration of the tumor exosomes in a system, the tumor exosome can be quantitatively detected through the fluorescence signal ratio of the fluorescent molecules FAM and TAMRA.
2. The method for detecting tumor exosomes identified by aptamer according to claim 1, wherein the hairpin probe H1 is formed by a hairpin structure formed by an aptamer sequence apt and a trigger sequence I of hybridization chain reaction through an extension sequence, the trigger sequence I is in a locked state due to intramolecular nucleic acid hybridization; after the aptamer sequence apt in the hairpin probe H1 is combined with tumor exosomes, the trigger sequence I required by hybridization chain reaction is released.
3. The aptamer-recognized tumor exosome detection method according to claim 1, wherein the sequences of the first signaling probe H2 and the second signaling probe H3 are partially complementary, and fluorescent molecules FAM and TAMRA capable of fluorescence resonance energy transfer are modified at the 3' end of the first signaling probe H2 and the ring portion of the second signaling probe H3 respectively.
4. The aptamer-recognized tumor exosome detection method according to claim 1, comprising the steps of:
(1) preparing a recognition probe: dissolving a probe containing the aptamer sequence apt and the trigger sequence I in 5 mM MgCl containing 250 nM glucose2·6H2In PBS buffer solution of O, the concentration of the probe is 400 nM, the solution is put on a vortex mixer for 30 s and mixed evenly, then the solution is put in a water bath, the temperature is gradually raised to 90 ℃, the solution is taken out after 10 min, the solution is slowly cooled to the room temperature, and a hairpin-shaped probe H1, namely a recognition probe, is obtained by utilizing the complementary hybridization reaction of nucleic acid;
(2) and preparing a signal probe: with 5 mM MgCl containing 250 nM glucose2·6H2Dissolving a first signal probe H2 modified by fluorescent molecule FAM and a second signal probe H3 modified by fluorescent molecule TAMRA in PBS buffer solution of O respectively, wherein the concentrations of the first signal probe H2 and the second signal probe H3 are both 800 nM, uniformly mixing the two in a vortex mixer for 30 s, then placing the mixture in a water bath, gradually raising the temperature to 90 ℃, taking out the mixture after 10 min, slowly cooling the mixture to room temperature, then respectively mixing the mixture with the same amount of the first signal probe H2 and the second signal probe H3, and uniformly mixing the mixture in the vortex mixer for 30 s to obtain signal probes;
(3) tumor exosome detection: adding the recognition probe and the signal probe prepared in the step into a cell solution, wherein the ratio of the concentration of the recognition probe to the concentration of the signal probe is 1:4, reacting for 3h, when no tumor exosome exists, the trigger sequence I in the recognition probe cannot trigger the hybridization chain reaction of the signal probe, at the moment, the FAM and the TAMRA are far away, the FRET signal between the FAM and the TAMRA cannot be detected, and when the tumor exosome exists, detecting the fluorescence emission intensity of the FAM and the TAMRA by using a fluorescence spectrophotometer under the excitation of 480 nm excitation light, so as to realize the quantitative detection of the tumor exosome.
5. The method for detecting tumor exosomes identified by aptamer according to any one of claims 1 to 4, wherein the tumor exosomes are lung cancer A549 cells.
6. The aptamer-recognized tumor exosome detection method according to claim 5, comprising the steps of:
(1) lung cancer A549 cell culture and exosome extraction: using an RPMI 1640 culture medium containing 10% by mass of exosome-free fetal bovine serum FBS as a cell culture solution, supplementing penicillin or streptomycin with the mass concentration of 1% into the culture solution, culturing lung cancer A549 cells in a carbon dioxide incubator with the volume concentration of 5%, wherein the temperature is 37 ℃, and collecting cell supernatant when the cells grow to 80-90%; separating and purifying the cell exosomes in the collected cell supernatant by using a centrifugal ultrafiltration method, which specifically comprises the following steps: centrifuging the collected cell supernatant for 30min under the centrifugation condition of 2500 g, filtering the supernatant with an ultrafiltration membrane of 0.22 m to remove cell debris, centrifuging the collected liquid with an ultrafiltration tube of 100 kDa for 30min under the condition of 5000 g, dissolving the collected solution containing exosomes in PBS of pH 7.4, and storing at-80 ℃ for later use;
(2) and preparing a recognition probe: dissolving a probe containing the aptamer sequence apt and the trigger sequence I in 5 mM MgCl containing 250 nM glucose2·6H2In PBS buffer solution of O, the concentration of the probe is 400 nM, the solution is put on a vortex mixer for 30 s and mixed evenly, then the solution is put in a water bath, the temperature is gradually raised to 90 ℃, the solution is taken out after 10 min, the solution is slowly cooled to the room temperature, and a hairpin-shaped probe H1, namely a recognition probe, is obtained by utilizing the complementary hybridization reaction of nucleic acid;
(3) and preparing a signal probe: with 5 mM MgCl containing 250 nM glucose2·6H2Dissolving a fluorescent molecule FAM modified first signal probe H2 and a fluorescent molecule TAMRA modified second signal probe H3 in a PBS buffer solution of O respectively, wherein the concentrations of the first signal probe H2 and the second signal probe H3 are both 800 nM, uniformly mixing the two on a vortex mixer for 30 s, then placing the mixture in a water bath, gradually raising the temperature to 90 ℃, taking out the mixture after 10 min, slowly cooling the mixture to room temperature, and then respectively mixing the mixture with the same amount of the first signal probe H2 and the second signal probe H3525Mixing a signal probe H2 with a second signal probe H3, and uniformly mixing on a vortex mixer for 30 s to obtain a signal probe;
(4) drawing a standard curve: respectively adding the prepared recognition probe and the prepared signal probe into exosomes derived from lung cancer A549 cells with different concentrations, wherein the concentration of the recognition probe and the concentration of the signal probe are respectively 50nM and 200nM, reacting for 3h, detecting the fluorescence emission intensity of FAM at 522 nM and TAMRA at 580nM under the excitation of 480 nM exciting light by using a fluorescence spectrophotometer, taking the concentration of the exosomes as an abscissa and the ratio of the fluorescence intensity at 522 nM and 580nM as an ordinate, and drawing a standard curve, wherein R is the ratio of the fluorescence intensity at the 580nM to the fluorescence intensity at the 580nM, and the standard curve is drawn, wherein R is the ratio of the fluorescence intensity at the corresponding position of the exosomes to the fluorescence intensity at the corresponding position of the exosomes, and the ratio of the fluorescence intensity at the corresponding position of the exosomes to the corresponding position of the FAM to the corresponding position of the lung cancer A549 cells2Is 0.992; it is found that the fluorescence intensity ratio is larger as the concentration of the lung cancer A549 cells is larger,
(5) selective response study:
culturing and collecting normal cell culture solution supernatant, respectively collecting and extracting exosomes by adopting the same method as the step (1), taking the obtained exosomes of different cell sources as a reference, enabling the total amount of the exosomes contained in each solution to be detected to be equivalent, adding a detection probe with the same concentration under the same condition, recording the fluorescence intensity value of each sample at the emission wavelength of fluorescent molecules FAM and TAMRA through a fluorescence instrument, and taking the ratio of the fluorescence intensity value to the total amount of the exosomes as a signal of the fluorescence resonance energy transfer degree of a system to obtain that the fluorescence resonance energy transfer signals generated by the exosomes of the reference cell source are weaker and far lower than the signal intensity generated by the exosomes of the lung cancer A549 cell source with the same concentration; the kit shows that the kit can specifically identify and detect the exosomes from the lung cancer A549 cells, does not respond to the exosomes from other different cell sources, and detects the high selectivity of the lung cancer A549 cell exosomes;
(6) and detecting exosomes in the complex biological sample: and (2) detecting fluorescence resonance energy signals of the system in PBS and 10% FBS respectively by taking the Bst cell exosomes as an interferent to obtain that the fluorescence signals of an exosome group containing a Bst cell source and a group without the interferent exosomes have no obvious difference, and the results in 10% FBS and the results in PBS both show interference, which indicates that the detection of the target tumor cell exosomes has no interference.
7. The method for detecting tumor exosomes identified by aptamer according to any one of claims 1 to 6, wherein the sequence of aptamer sequence apt is: 5'-TTT ATG GGT GGG TGG GGG GTT TTT-3';
the sequence of trigger sequence I is: 5'-CACCCATAAAGACTGATGTTGA-3', respectively;
the sequence of hairpin probe H1 is: 5'-CAC CCA TAA AGA CTG ATG TTG ATC AGT CTT TAT GGG TGG G GGG GGG TTT TTT T-3', respectively;
the sequence of the first signal probe H2 for modifying the fluorescent molecule FAM is as follows:
5’- TCA ACA TCA GTC TTT ATG GGT GCA AGC ACC CAT AAA GAC TGA -3’-FAM；
the sequence of the second signal probe H3 for modifying the fluorescent molecule TAMRA is as follows:
5’- C ACC CAT AAA GAC TGA TGT TGA T（TAMRA）CA GTC TTT ATG GGT GCT TG -3’。
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