Probe composition for detecting single exosome subgroup, CRISPR / Cas12a reporter as well as preparation and application of probe composition and CRISPR / Cas12a reporter

Abstract

The invention belongs to the technical field of biology, and particularly discloses a probe composition for detecting a single exosome subgroup, a CRISPR / Cas12a reporter and preparation and application of the probe composition and the CRISPR / Cas12a reporter. The probe composition comprises two aptamer probes, after target proteins (PD-L1 and EpCAM) on an exosome membrane are recognized, allosteric reaction occurs, a specific binding region with crRNA is exposed, the aptamer probes are hybridized with Cas12a / crRNA, a Cas12a trans-cleavage report chain is synergistically activated, and a fluorescence signal is generated; the intensity of the fluorescence signal is in direct proportion to the number of exosomes, so that the exosome subgroups can be directly quantified; a reaction system is limited in liquid drops by virtue of a liquid drop micro-fluidic technology, and the detection sensitivity can be improved by virtue of a dual confinement effect. Based on this, the invention develops a CRISPR / Cas12a reporter, a system, a method and the like for detecting a single exosome subgroup, the operation process can be simplified, and one-step detection is realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a probe composition for detecting a single exosome subpopulation, a CRISPR / Cas12a reporter, and preparation and application thereof. Background Art

[0002] Exosomes (Exo) are extracellular vesicles (EVs) produced by eukaryotic cells, reflecting changes in the physiological and pathological states of parental cells. As messengers of intercellular communication, they are widely involved in cancer metastasis, immune response, and tissue repair. Exo show complex heterogeneity in molecular characteristics, originating from the generation of multiple exosome subpopulations with different structures and compositions. In particular, tumor-derived exosomes (tExo) subpopulations can promote tumorigenesis and tumor progression, and have great prospects as ideal non-invasive biomarkers for cancer screening and treatment monitoring. However, due to complex background interference, heterogeneous Exo subtypes, and different exosomal protein expression, the precise identification of tExo subpopulations remains a huge challenge. Currently, ultracentrifugation is the most commonly used technique for separating Exo, but it is inefficient and cannot quantitatively separate Exo subpopulations. Most other types of detection methods often involve complex purification steps and dedicated equipment, which are not suitable for efficient operation in scientific research and clinical applications; moreover, most analytical methods can only be used to identify a single biomarker of EVs, which also reduces the specificity of EVs analysis, which hinders the clinical application of EVs in cancer diagnosis. Summary of the invention

[0003] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a probe composition for detecting a single exosome subpopulation, a CRISPR / Cas12a reporter, and its preparation and application, so as to solve the current difficulties in analyzing exosome heterogeneity and the complexity of detection methods.

[0004] To achieve the above-mentioned and other related purposes, the present invention provides a probe composition, comprising a first aptamer probe (i.e., H P probe) and the second aptamer probe (i.e., H E probe);

[0005] The nucleotide sequence of the first aptamer probe includes a first aptamer region and a first complementary region, wherein the first aptamer region includes an aptamer that can specifically bind to programmed cell death ligand 1 (PD-L1) protein on the Exo membrane, and the first complementary region is complementary to a partial crRNA sequence;

[0006] The nucleotide sequence of the second aptamer probe includes a second aptamer region and a second complementary region, the second aptamer region includes an aptamer that can specifically bind to the epithelial cell adhesion molecule (EpCAM) protein on the Exo membrane, and the second complementary region is complementary to the crRNA partial sequence;

[0007] The nucleotide sequences of the first probe and the second probe are highly non-complementary.

[0008] In some embodiments, the aptamer that can specifically bind to the PD-L1 protein on the Exo membrane is an MJ5C aptamer.

[0009] In some embodiments, the aptamer that can specifically bind to the EpCAM protein on the Exo membrane is a SYL3C aptamer.

[0010] In some embodiments, the first aptamer region is located at the head of the first aptamer probe.

[0011] In some embodiments, the second aptamer region is located at the tail of the second aptamer probe.

[0012] In some embodiments, the nucleotide sequence of the crRNA is as shown in SEQ ID NO.1.

[0013] In some embodiments, the first complementary region is T1, and the nucleotide sequence of T1 is shown in SEQ ID NO.2.

[0014] In some embodiments, the second complementary region is T2, and the nucleotide sequence of T2 is shown in SEQ ID NO.3.

[0015] In some embodiments, the nucleotide sequence of the first aptamer probe further includes a first spacer region between the first aptamer region and the first complementary region, and the first spacer region is a repeating sequence containing 10 to 40 T bases.

[0016] In some embodiments, the nucleotide sequence of the second aptamer probe further includes a second spacer region between the second aptamer region and the second complementary region, and the second spacer region is a repeating sequence containing 10 to 40 base Ts.

[0017] In some embodiments, the nucleotide sequence of the first aptamer probe is shown as SEQ ID NO.4.

[0018] In some embodiments, the nucleotide sequence of the second aptamer probe is shown as SEQ ID NO.5.

[0019] In some embodiments, the first aptamer probe further includes a fluorescent group and / or a quencher group.

[0020] In some embodiments, the second aptamer probe further comprises a fluorescent group and / or a quencher group.

[0021] In some embodiments, the fluorescent group includes but is not limited to 6-FAM and the like, and the quenching group includes but is not limited to BHQ1 and the like.

[0022] The present invention also provides a CRISPR / Cas12a reporter for detecting a single exosome subpopulation, comprising a reaction system, the reaction system comprising the probe composition as described above, and further comprising a target exosome, a Cas12a / crRNA complex (Cas12a / crRNA complex) and a reporter strand (FHQ); the Cas12a / crRNA complex is formed by connecting a Cas12a enzyme to a crRNA;

[0023] After the first aptamer probe and the second aptamer probe recognize the target protein on the exosome (Exo) membrane, conformational changes occur, exposing the first complementary region and the second complementary region; the first complementary region and the second complementary region specifically bind to the crRNA through oligonucleotide hybridization, synergistically activate the trans-cleavage activity of the Cas12a enzyme, cut the reporter chain (FHQ), and generate a fluorescent signal.

[0024] In some embodiments, the reaction system is a homogeneous system, including the probe composition as described above, and further including target exosomes, Cas12a enzyme, crRNA and reporter chain (FHQ).

[0025] In some embodiments, the reaction system is a droplet system, the droplet system includes aqueous phase 1 and aqueous phase 2, the aqueous phase 1 includes Cas12a enzyme, crRNA and reporter chain (FHQ), the aqueous phase 2 includes the probe composition as described above, and also includes target exosomes; the aqueous phase 1, the aqueous phase 2 and the droplet generation oil jointly generate droplets. Wherein, the droplet generation oil is a droplet generation oil commonly used in droplet microfluidics technology, including but not limited to mineral oil, isopropyl palmitate, Drop-Surf fluorine oil, etc.

[0026] In some embodiments, in the reaction system, the molar contents of the first aptamer probe, the second aptamer probe, the Cas12a enzyme, the crRNA and the reporter chain (FHQ) are the same.

[0027] In some embodiments, the nucleotide sequence of the reporter chain (FHQ) is shown as SEQ ID NO.7.

[0028] In some embodiments, the reporter chain (FHQ) further comprises a fluorescent group and / or a quencher group, wherein the fluorescent group includes but is not limited to 6-FAM and the like, and the quencher group includes but is not limited to BHQ1 and the like.

[0029] In some embodiments, the reaction system further comprises a buffer, a solvent, etc. The buffer includes but is not limited to PBS buffer, NE buffer, etc., and the solvent includes but is not limited to ultrapure water, deionized water, etc.

[0030] The present invention also provides a method for preparing a CRISPR / Cas12a reporter for detecting a single exosome subpopulation as described above, comprising:

[0031] When the reaction system is a homogeneous system, preparing the homogeneous system and performing an incubation reaction;

[0032] And / or, when the reaction system is a droplet system, aqueous phase 1 and aqueous phase 2 are prepared separately, introduced into a microfluidic chip separately, and droplets are generated together with droplet generating oil, and the generated droplets are incubated for reaction.

[0033] In some embodiments, the incubation reaction temperature is 37°C.

[0034] In some embodiments, the incubation reaction time is 30 min.

[0035] In some embodiments, before preparing the reaction system, the first aptamer probe, the second aptamer probe, the crRNA and the reporter strand (FHQ) are denatured and annealed. In a specific embodiment, the denaturation temperature is 95° C. and the denaturation time is 5 min.

[0036] The present invention also provides the use of the probe composition as described above, the CRISPR / Cas12a reporter as described above and / or the CRISPR / Cas12a reporter prepared by the method as described above in the preparation of a single exosome subpopulation detection reagent and / or a tumor diagnostic reagent.

[0037] In some embodiments, the exosomes are tumor-derived exosomes.

[0038] The present invention also provides a system for detecting a single exosome subpopulation, comprising the probe composition as described above, the CRISPR / Cas12a reporter as described above, and / or the CRISPR / Cas12a reporter prepared by the method as described above, and also comprising at least one of the following detection equipment: a fluorescence spectrophotometer, a microfluidic droplet generator, and a fluorescence microscope, but not limited thereto.

[0039] The present invention also provides a method for detecting a single exosome subpopulation for non-disease diagnosis or treatment purposes, comprising using the probe composition as described above, the CRISPR / Cas12a reporter as described above, and / or the CRISPR / Cas12a reporter prepared by the method as described above.

[0040] In some embodiments, the detection method comprises:

[0041] When the reaction system is a homogeneous system, preparing the homogeneous system, performing an incubation reaction, and obtaining a reactant; detecting a fluorescent signal of the obtained reactant, and detecting a single exosome subpopulation based on the detected fluorescent signal;

[0042] And / or, when the reaction system is a droplet system, aqueous phase 1 and aqueous phase 2 are prepared separately, aqueous phase 1 and aqueous phase 2 are introduced into a microfluidic chip respectively, droplets are generated together with droplet generating oil, the generated droplets are incubated for reaction, and then placed under a fluorescence microscope for imaging, and a single exosome subpopulation is detected based on the imaging results.

[0043] In some embodiments, the incubation reaction temperature is 37°C.

[0044] In some embodiments, the incubation reaction time is 30 min.

[0045] In some embodiments, before preparing the reaction system, the first aptamer probe, the second aptamer probe, the reporter strand (FHQ) and the crRNA are denatured and annealed. In a specific embodiment, the denaturation temperature is 95° C. and the denaturation time is 5 min.

[0046] As described above, the probe composition for detecting a single exosome subpopulation, the CRISPR / Cas12a reporter and the preparation and application thereof of the present invention have the following beneficial effects:

[0047] Based on two protein-activated aptamer probes and a dual-target activated CRISPR / Cas12a system, the present invention constructs a CRISPR / Cas12a reporter with good fluid and spatial dual confinement, and combines it with droplet microfluidics to develop a rapid and highly sensitive detection strategy, which can ultimately convert the protein signal on the Exo membrane surface into a fluorescent signal for output, thereby realizing direct detection of a single tExo subpopulation.

[0048] (1) The probe designed in the present invention can specifically bind to the CRISPR / Cas12a system, utilize the trans-cleavage activity of the Cas12a enzyme to generate a fluorescent signal, and finally convert the protein signal on the surface of the tExo membrane into a fluorescent signal for output.

[0049] (2) The dual confinement effect of fluid and space in the present invention significantly enhances the fluorescence signal of the reporter chain and improves the sensitivity of target Exo detection.

[0050] (3) In existing exosome detection technologies, many exosome detections require washing to remove unbound probes, but the method of the present invention does not require washing, which greatly simplifies the operation process and realizes one-step detection.

[0051] (4) The present invention utilizes aptamer probes to identify PD-L1 and EpCAM on the surface of Exo membranes. Compared with antibodies, aptamers can be modified through simple designs, and their smaller steric hindrance can effectively identify target Exo.

[0052] (5) The present invention integrates the CRISPR / Cas12a system and droplet microfluidics technology, which can be directly quantified according to the Poisson distribution. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Schematic diagram of a fluid and spatially dual-confined CRISPR / Cas12a reporter for detecting a single exosome subpopulation in an embodiment of the present invention.

[0054] Figure 2 Schematic diagram (A) and the obtained fluorescence signal diagram (B) for verifying the feasibility of the FASTER system probe conformational change in Example 2 of the present invention.

[0055] Figure 3 Schematic diagram (A) and the obtained fluorescence spectrum (B) for verifying the feasibility of synergistic activation of FASTER system probes in Example 3 of the present invention.

[0056] Figure 4 The fluorescence signal diagram (A) and droplet imaging diagram (B) obtained for verifying the specificity of the FASTER system in Example 5 of the present invention.

[0057] Figure 5 The fluorescence signal diagram (A) and the droplet signal diagram (B) obtained by verifying the sensitivity of the FASTER system in Example 6 of the present invention.

[0058] Figure 6 This is a droplet imaging image obtained by the FASTER system in Example 7 of the present invention to directly detect the feasibility of exosomes in clinical samples. DETAILED DESCRIPTION

[0059] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0060] In the present invention, unless otherwise specified, the term "plurality" means two or more.

[0061] The character " / " indicates that the objects before and after it are in an "or" relationship. For example, A / B means: A or B.

[0062] The term "and / or" is a description of the association relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or, A and B.

[0063] At present, many tumor-related protein biomarkers have been identified in Exo from blood samples, and their types and expression levels are closely related to the occurrence and progression of cancer. For example, PD-L1 is a key immune checkpoint protein that mediates tumor cell immunosuppression. It has been confirmed that EVs of tumor subtypes contain a large amount of membrane protein PD-L1, which can directly inhibit the anti-tumor activity of effector CD8+T cells and NK cells, thereby promoting tumor cells to escape the surveillance of the immune system. In addition, EpCAM is involved in a variety of cancer processes, plays an important role in tumor invasion and spread, is highly expressed in most epithelial cancers, and is currently a target for molecular therapy.

[0064] Aptamers are functional oligonucleotides obtained through in vitro or in vivo screening, with unique structures, high specificity and affinity. Compared with antibodies, they have the advantages of small size, good stability, and flexible chemical modification. By designing aptamer probes for different proteins, Exo surface proteins can be effectively identified without complex DNA-antibody coupling.

[0065] On this basis, the present invention designs a fluidic and spatially dual-confined CRISPR / Cas12a reporter (Fluidically and Spatially Dual-Confined Cas12a Reporter, FASTER) for detecting a single tExo subpopulation. With the help of droplet microfluidics technology, droplets containing 0 or 1 target Exo are generated; in the droplets containing target Exo, the aptamer probe adds molecular markers on the Exo membrane proteins PD-L1 and EpCAM, and then the conformationally modified aptamer probe synergistically activates Cas12a / crRNA, and trans-cuts the substrate to produce a fluorescent signal. Thus, a method for rapid and sensitive detection of a single tExo subpopulation is constructed. This strategy uses the method of simultaneously identifying two targets to synergistically activate the reaction system to enhance the ability to distinguish Exo subtypes, and can directly quantify Exo, thereby distinguishing tumor patients from normal people. This strategy provides ideas for the analysis of a single exosome subpopulation, and also promotes the clinical application of Exo as a liquid biopsy target.

[0066] like Figure 1 As shown, in one embodiment of the present invention, two highly non-complementary specific recognition probes are designed: one is H with the head of the MJ5C aptamer. P The probe (PD-L1 probe) can specifically recognize and bind to the PD-L1 protein on the Exo membrane; the other is the H E The probe (EpCAM probe) can specifically recognize and bind to the EpCAM protein on the Exo membrane. The two probes contain three regions, namely the aptamer region, the spacer region, and the region that specifically binds to crRNA. The region that specifically binds to crRNA refers to the region that is complementary to the partial sequence of crRNA. Droplet microfluidics technology is used to generate droplets containing 0 or 1 target Exo. In the droplet containing the target Exo, when the probe recognizes the target protein on the Exo membrane, it undergoes conformational changes, exposing the region that specifically binds to crRNA; H P and H EThrough oligonucleotide hybridization and binding with Cas12a / crRNA, Cas12a / crRNA is synergistically activated, and the trans-cleavage activity of Cas12a is activated, which can trans-cleave the reporter chain FHQ (Hairpin reporter) to generate a fluorescent signal. This design uses two specific recognition elements to effectively ensure the specificity of detecting Exo subpopulations; only Exo that express PD-L1 protein and EpCAM at the same time can be effectively captured, and then synergistically activate Cas12a / crRNA to generate a fluorescent signal. The intensity of the fluorescent signal is proportional to the number of Exo. According to the Poisson distribution, the Exo subpopulation can be directly quantified. This strategy uses Cas12a / crRNA with the help of H P and H E The probe is anchored on the Exo fluid membrane by hybridization; at the same time, with the help of droplet microfluidics technology, the entire reaction system is confined within the droplet; the double confinement effect greatly improves the detection sensitivity.

[0067] The following specific examples are given to illustrate the present invention in detail. It should also be understood that the following examples are only used to specifically illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range according to the description herein, and are not limited to the specific values ​​​​exemplified below.

[0068] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0069] 1. Experimental Materials

[0070] All oligonucleotides purified by HPLC, DEPC water, and PBS were purchased from Sangon Biotechnology Co., Ltd (Shanghai, China). LbCas12a enzyme and 10×NEBuffer r2.1 were purchased from New England Biolabs (USA). Droplet generation oil was purchased from Bio-Rad (USA). All chemical reagents were of analytical grade and used directly without further purification.

[0071] 2. Experimental Instruments

[0072] Fluorescence spectra were measured on a F-4700 fluorescence spectrophotometer (Hitachi, Japan). Fluorescence imaging of droplets was performed on a TCS SP8 confocal laser scanning microscope (Leica, Germany).

[0073] The nucleotide sequence table involved in the embodiments of the present invention is shown in Table 1.

[0074] Table 1 Nucleotide sequence table

[0075]

[0076] Example 1

[0077] The implementation process of this embodiment is as follows:

[0078] 1. Extraction of exosomes

[0079] Cell-derived exosomes were extracted by ultracentrifugation. The supernatant was collected after 48 h of cell starvation culture, and the precipitate was discarded after low-speed centrifugation (2000g, 30min, 4°C); the precipitate was discarded after centrifugation (10,000g, 30min, 4°C), and the supernatant was filtered with a 0.22μm filter to remove cells and apoptotic debris in the serum. In order to remove proteins and larger extracellular vesicles, ultracentrifugation (110,000g, 70min, 4°C) was performed, the supernatant was discarded, and after resuspending with PBS, ultracentrifugation was repeated twice to obtain the exosome sediment. Finally, the precipitated exosomes were resuspended in 100μL PBS and stored at -80°C until use.

[0080] Serum samples were collected for 1 mL and centrifuged at low speed (3000 g, 10 min, 4 ° C) to obtain free serum. Then, the serum was centrifuged again (10,000 g, 20 min, 4 ° C) to remove large vesicles. The serum samples were filtered with a 0.22 μm filter and stored at -80 ° C for further use. Serum samples (10 μL) were taken for FASTER detection.

[0081] 2. Construction of FASTER reaction system:

[0082] According to the following steps, a FASTER reaction system (homogeneous system and droplet system) was constructed according to Table 2, and the reaction system was reacted at 37° C. for 30 min.

[0083] (a) Probe H P and H E Combining exosomal PD-L1 and EpCAM: First, P , H E , FHQ and crRNA were dissolved in DEPC water, followed by denaturation (95°C, 5 min) and slow annealing steps.

[0084] (b) preparing a homogeneous reaction system and reacting at 37° C. for 30 min;

[0085] (c) The droplet system is configured with aqueous phase 1 and aqueous phase 2 respectively.

[0086] Table 2 FASTER reaction system (homogeneous system and droplet system)

[0087] Element concentration volume Cas12a 1μM 2μL crRNA 1μM 2μL NEBuffer r2.1 10× 10μL FWf 1μM 10μL <![CDATA[H E ]]> 1μM 2μL <![CDATA[H P ]]> 1μM 2μL Exo 10μL DEPC 62μL

[0088]

[0089]

[0090] 3. Add the above homogeneous system reactants into a quartz cuvette and measure using a fluorescence spectrophotometer, which specifically includes the following steps:

[0091] (1) Fluorescence cuvette cleaning: Soak the fluorescence cuvette in alcohol and clean it with DEPC water;

[0092] (2) Setting parameters: set the excitation wavelength to 492 nm, the emission wavelength range to 500-600 nm, and the voltage to 600 V;

[0093] (3) Perform detection: Add the reaction solution into the fluorescence cuvette and click on “Detect” to obtain the fluorescence signal.

[0094] 4. Introduce the reactants of the above droplet system into the microfluidic chip and use a fluorescence microscope for imaging. The specific steps are as follows:

[0095] (1) introducing aqueous phase 1 and aqueous phase 2 into two chip inlets respectively to generate droplets together with droplet generating oil;

[0096] (2) incubating the generated droplets at 37°C for 30 min;

[0097] (3) Place the droplet under a fluorescence microscope for imaging.

[0098] Example 2

[0099] This example verifies the feasibility of probe conformational change, and the implementation process is as follows:

[0100] For H in Example 1 P and H E The conformational changes were verified by fluorescence signal, and the results were as follows Figure 2 As shown, Figure 2 A is a schematic diagram of conformational change, and four groups of experiments were set up:

[0101] 1. Probes labeled with FAM and BHQ1 groups (QH P -F and FH E -Q) and exosomes

[0102] 2. Probes labeled with only FAM groups (H P -F and FH E )

[0103] 3. Probes labeled with FAM and BHQ1 groups (QH P -F and FH E -Q)

[0104] 4. Exosomes

[0105] like Figure 2 As shown in B, QH P -F and FH E -Q fluorescence signal compared to H P -F and FH E The significant decrease indicates the successful blocking of the probe; QH P -F and FH E -Q and exosomes group compared with only QH P -F and FH E For the group with only exosomes or the group with only Q, the fluorescence signal was significantly restored, which was different from that of the group with H P -F and FH E The fluorescence signal of the probe was close to that of the target protein on the exosome membrane, indicating that the probe successfully underwent conformational change after recognizing the target protein on the exosome membrane.

[0106] Example 3

[0107] This example verifies the feasibility of probe cooperative activation, and the implementation process is as follows:

[0108] like Figure 3 As shown in A, H P and H E The specific binding crRNA regions T1 and T2 contained in the PCR product were used as crRNA targets to verify the ability of T1 and T2 to synergistically activate Cas12a trans-cleavage. Figure 3 As shown in B, four groups of experiments were set up:

[0109] 1. T1 and T2

[0110] 2. T1

[0111] 3. T2

[0112] 4. No target

[0113] The experimental results show that only when T1 and T2 exist at the same time can they hybridize with crRNA, activate the trans-cleavage activity of Cas12a, and generate fluorescent signals. However, when T1 or T2 exists alone, no fluorescent signals can be generated, indicating that T1 and T2 can synergistically activate Cas12a / crRNA.

[0114] Example 4

[0115] This example verifies the specificity of the FASTER system, and the implementation process is as follows:

[0116] Exosomes from three different types of breast cancer cells were selected: TNBC cell line (MDA-MB-231), HER2 cell line (SK-BR-3), luminal cell line (MCF-7) and a normal breast cell line (MCF-10A). MDA-MB-231 (PD-L1 + EpCAM + )、Exo SK-BR-3 (PD-L1 + EpCAM + )、Exo MCF-7 (PD-L1 + EpCAM + )、Exo MCF-10A (PD-L1 + EpCAM - ) to verify the specificity of FASTER in recognizing target proteins. Eight groups of experiments were set up on four types of exosomes according to the probes and exosomes, namely:

[0117] 1. Exo+H E +H P

[0118] 2. H E

[0119] 3. H P

[0120] 4. H E +H P

[0121] 5. Exo

[0122] 6. Exo+H E

[0123] 7. Exo+H P

[0124] 8. No Exo and H E , H P

[0125] like Figure 4 As shown in A and 4B, only Exo, H E , H P Only the group with both PD-L1 and EpCAM proteins present and expressed on the Exo membrane will output fluorescent signals, indicating the high specificity of FASTER in detecting exosomes.

[0126] Example 5

[0127] This example verifies the sensitivity of the FASTER system, and the implementation process is as follows:

[0128] According to Table 2, the FASTER homogeneous reaction system was constructed, and four exosomes with different concentration gradients were added. MDA-MB-231 (PD-L1 + EpCAM + )、Exo SK-BR-3 (PD-L1 + EpCAM + )、Exo MCF-7 (PD-L1 + EpCAM + )、Exo MCF-10A (PD-L1 + EpCAM - ), and the above systems were incubated at 37°C for 30 min and then the fluorescence signals were detected.

[0129] like Figure 5 As shown in A, in Exo showing fluorescent signal MDA-MB-231 、Exo SK-BR-3 、Exo MCF-7 The linear range of the homogeneous system is 1×10 3 ~1×10 8 particles / μL, and the minimum detection limit is 1×10 3 particles / μL; e.g. Figure 5 As shown in B, in Exo showing fluorescent signal MDA-MB-231 、Exo SK-BR-3 、Exo MCF-7 The linear range of the droplet system is 1×10 1 ~1×10 6 particles / μL, and the minimum detection limit is 1×10 1 particles / μL, demonstrating the high sensitivity of the FASTER system in detecting exosomes.

[0130] Example 6

[0131] This example verifies the feasibility of the FASTER system to directly detect exosomes in clinical samples. The implementation process is as follows:

[0132] 1 mL of serum sample from cancer patients was taken and centrifuged at low speed (3000 g, 10 min, 4 ° C) to obtain free serum. Then, the serum was centrifuged again (10,000 g, 20 min, 4 ° C) to remove large vesicles. The serum sample was filtered with a 0.22 μm filter, and the serum sample (10 μL) was prepared according to Table 2. Water phase 1 and water phase 2 were respectively prepared, and droplets were generated using droplet microfluidics technology. After incubation at 37 ° C for 30 min, imaging was performed.

[0133] like Figure 6 As shown, compared with the blank control group (no target) and the healthy control group (No. 1-19), the serum samples of cancer patients (No. 20-39) showed obvious fluorescence signals, indicating that the FASTER system can directly detect exosomes in clinical samples.

[0134] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A probe composition, characterized in that comprising a first aptamer probe and a second aptamer probe; The nucleotide sequence of the first aptamer probe includes a first aptamer region and a first complementary region, the first aptamer region includes an aptamer that can specifically bind to the programmed cell death ligand 1 protein on the exosome membrane, and the first complementary region is complementary to a partial crRNA sequence; The nucleotide sequence of the second aptamer probe includes a second aptamer region and a second complementary region, the second aptamer region includes an aptamer that can specifically bind to an epithelial cell adhesion molecule protein on an exosome membrane, and the second complementary region is complementary to a partial crRNA sequence; The nucleotide sequences of the first probe and the second probe are highly non-complementary.

2. The probe composition according to claim 1, characterized in that: The aptamer that can specifically bind to the PD-L1 protein on the exosome membrane is an MJ5C aptamer; And / or, the aptamer that can specifically bind to the EpCAM protein on the Exo membrane is a SYL3C aptamer; and / or, the first aptamer region is located at the head of the first aptamer probe; and / or, the second aptamer region is located at the tail of the second aptamer probe; And / or, the nucleotide sequence of the crRNA is shown in SEQ ID NO.1; And / or, the first complementary region is T1, and the nucleotide sequence of T1 is shown in SEQ ID NO.2; And / or, the second complementary region is T2, and the nucleotide sequence of T2 is shown in SEQ ID NO.3; And / or, the nucleotide sequence of the first aptamer probe further includes a first spacer region between the first aptamer region and the first complementary region, and the first spacer region is a repeating sequence containing 10 to 40 base Ts; And / or, the nucleotide sequence of the second aptamer probe further includes a second spacer region between the second aptamer region and the second complementary region, and the second spacer region is a repeating sequence containing 10 to 40 base Ts.

3. The probe composition according to claim 2, characterized in that: The nucleotide sequence of the first aptamer probe is shown in SEQ ID NO.4; And / or, the nucleotide sequence of the second aptamer probe is as shown in SEQ ID NO.

5.

4. The probe composition according to any one of claims 1 to 3, characterized in that: The first aptamer probe further includes a fluorescent group and / or a quenching group; And / or, the second aptamer probe further includes a fluorescent group and / or a quenching group.

5. A CRISPR / Cas12a reporter for detecting a single exosome subpopulation, characterized in that: Comprising a reaction system, the reaction system comprising the probe composition according to any one of claims 1 to 4, and further comprising a target exosome, a Cas12a / crRNA complex and a reporter strand; the Cas12a / crRNA complex is formed by connecting a Cas12a enzyme to a crRNA; After the first aptamer probe and the second aptamer probe recognize the target protein on the Exo membrane, conformational changes occur, exposing the first complementary region and the second complementary region; the first complementary region and the second complementary region specifically bind to crRNA through oligonucleotide hybridization, synergistically activate the trans-cleavage activity of the Cas12a enzyme, cut the reporter chain, and generate a fluorescent signal.

6. The CRISPR / Cas12a reporter according to claim 5, characterized in that: The reaction system is a homogeneous system, comprising the probe composition according to any one of claims 1 to 4, and further comprising target exosomes, Cas12a enzyme, crRNA and a reporter strand; And / or, the reaction system is a droplet system, the droplet system comprises an aqueous phase 1 and an aqueous phase 2, the aqueous phase 1 comprises a Cas12a enzyme, crRNA and a reporter chain, the aqueous phase 2 comprises the probe composition according to any one of claims 1 to 4, and further comprises target exosomes; the aqueous phase 1, the aqueous phase 2 and the droplet generating oil jointly generate droplets; And / or, the nucleotide sequence of the reporter chain is shown in SEQ ID NO.

7.

7. A method for preparing a CRISPR / Cas12a reporter for detecting a single exosome subpopulation according to any one of claims 5 to 6, comprising: When the reaction system is a homogeneous system, preparing the homogeneous system and performing an incubation reaction; And / or, when the reaction system is a droplet system, aqueous phase 1 and aqueous phase 2 are prepared separately, introduced into a microfluidic chip separately, and droplets are generated together with droplet generating oil, and the generated droplets are incubated for reaction.

8. Use of the probe composition according to any one of claims 1 to 4, the CRISPR / Cas12a reporter according to any one of claims 5 to 6, and / or the CRISPR / Cas12a reporter prepared by the method according to claim 7 in the preparation of a single exosome subpopulation detection reagent and / or a tumor diagnostic reagent.

9. A system for detecting a single exosome subpopulation, characterized in that: It comprises the probe composition as described in any one of claims 1 to 4, the CRISPR / Cas12a reporter as described in any one of claims 5 to 6, and / or the CRISPR / Cas12a reporter prepared by the method as described in claim 7, and also comprises at least one of the following detection equipment: a fluorescence spectrophotometer, a microfluidic droplet generator and a fluorescence microscope.

10. A method for detecting a single exosome subpopulation for non-disease diagnosis or treatment purposes, characterized in that: It includes a CRISPR / Cas12a reporter prepared using the probe composition as described in any one of claims 1 to 4, the CRISPR / Cas12a reporter as described in any one of claims 5 to 6, and / or the method as described in claim 7.

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