CN110082531B - Tumor exosome nano fluorescence detection kit and application thereof - Google Patents

Abstract

The invention provides a tumor exosome nano fluorescence detection kit and application thereof. The invention not only proves that PSMA on the exosome derived from human plasma can be used as a biomarker for identifying healthy subjects and prostate cancer patients, but also further proves that the method can effectively avoid the influence of the exosome derived from normal cells in a clinical plasma sample on the detection result, has good anti-interference capability and has great clinical application potential in the aspect of prostate cancer PSMA (+) exosome subgroup analysis.

Description

Tumor exosome nano fluorescence detection kit and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a Turn-on tumor exosome nano fluorescence detection kit based on an aptamer and an aggregation-induced emission material and application thereof.

Background

In recent years, the incidence of prostate cancer in China is increased year by year, the proportion of middle and late stages of newly diagnosed patients in most regions is higher than that in Europe and America, and the prostate cancer has the characteristics of rapid development and easy metastasis, can be diagnosed and treated in time before symptoms and signs appear, can obviously improve prognosis, and prolong the life cycle of patients. Therefore, the establishment of a sensitive, specific, noninvasive, convenient and rapid detection method for early screening, diagnosis or monitoring of the prostate cancer is particularly important for controlling the development of the prostate cancer and improving the quality of life of patients.

Liquid Biopsy (Liquid Biopsy) obtains Tumor information in body fluids by non-invasive sampling, mainly including Circulating Tumor Cells (CTCs), Circulating Tumor dna (ctdna), and exosomes (exosomes) with tumors or metastases released into the extracellular environment, to aid in cancer diagnosis and treatment, and is a representative diagnostic technique for "precision medicine". Compared with the traditional tissue biopsy, the liquid biopsy has the advantages of rapidness, convenience, small damage and the like. The diagnosis time of cancer is greatly shortened, and the treatment condition of cancer patients can be tracked at any time, so that individual accurate treatment is really realized. Therefore, the liquid biopsy is expected to become an ideal method for screening, diagnosing or monitoring early-stage prostate cancer.

Previous studies have demonstrated that exosomes, one of the liquid biopsies "sanjia horses", have a certain correlation with the occurrence, development, metastasis and drug resistance of prostate cancer, and are considered as prostate cancer liquid biopsy biomarkers with broad application prospects. The biosensing technology integrates the advantages of high efficiency, sensitivity, specificity, smallness, economy and the like, and is an important technical platform for realizing sensitive, specific, noninvasive, convenient and quick prostate cancer liquid biopsy. With the research of the nano technology being deepened in recent years, the nano material is gradually introduced into the design and application of the biosensing technology, and the nano fluorescence biosensor designed on the basis of the nano fluorescence biosensor gradually attracts people's attention due to the advantages of strong anti-interference performance, no need of a reference device, small sample amount, simple and convenient operation and the like, so that the construction of the exosome rapid detection method which is simple and convenient to operate, has high sensitivity and specificity and is an urgent need of prostate cancer liquid biopsy.

Disclosure of Invention

In view of the above disadvantages of the prior art, the present invention aims to provide a tumor exosome nano fluorescence detection kit, which is used for solving the problems of complex operation, long time consumption, weak anti-interference performance and the like of the detection method for prostate cancer in the prior art.

In order to achieve the above objects and other related objects, the present invention provides a kit for tumor exosome nano-fluorescence detection, comprising a recognition element, a fluorescence reporter group and a fluorescence quencher group.

Optionally, the recognition element is selected from a nucleic acid aptamer.

Optionally, the nucleotide sequence of the aptamer comprises any one of the sequences shown as SEQ ID NO.1-SEQ ID NO. 4.

Optionally, the fluorescent reporter group is selected from aggregation-induced emission molecules (AIE molecules).

Alternatively, the aggregation inducing luminescent molecule is selected from TTAPE (a derivative of tetraphenylethylene).

Optionally, the TTAPE is selected from TPE-TA (tertiary amine containing tetraphenylethylene), [12]]aneN₃modified tetraphenylethene(TPE)compounds([12]aneN₃A modified Tetraphenylethylene (TPE) compound derived from DOI: 10.1021/acsami.6b01949), TPE-Py molecule (derived from DOI: 10.1021/acs. analchem.5b04756), TPE-Z molecules (fromDOI：10.1016/j.talanta.2017.02.064)。

The above-mentioned AIE molecules are only partially exemplified, as long as AIE molecules capable of binding to DNA are suitable for the present invention.

Optionally, the fluorescence quenching group is selected from graphene oxide.

Alternatively, the graphene oxide is selected from single-layer graphene oxide, has a thickness of 0.8-1.2nm, is commercially available, and can be in the form of a sheet, a powder, a dispersion, preferably a powder.

The single-layer graphene oxide is in a flake shape, and the flake diameter of the powder is 500nm-5 mu m.

Optionally, a buffer is also included.

Optionally, the buffer is selected from 1X PBS buffer.

Optionally, the concentration of the fluorescent reporter group is 0.4 μ M, which is the system concentration.

Optionally, the volume of the fluorescent reporter is 0.5 μ L.

Optionally, the concentration of the fluorescence quenching group is 1mg/mL, which is the system concentration.

Optionally, the volume of the fluorescence quenching group is 2 μ L.

Optionally, the volume of the buffer is 48.5 μ L.

Optionally, the total volume of the kit is 60 μ L.

Optionally, the recognition element is at a concentration of 10 μ M and in a volume of 6 μ L.

The exosome targeted by the kit can be derived from various prostate cancer cells, including but not limited to LNCaP, PC3 and DU145 cells, and specifically can be plasma-derived exosomes of prostate cancer patients.

The exosome concentration may be 4.07 x 10⁵-1.83×10⁷particles/. mu.L in a volume of 3. mu.L.

The invention also provides application of the fluorescence detection kit in preparation or screening of prostatic cancer in preparation of a medicament for diagnosing and/or preventing and/or treating prostatic cancer, and the kit is used for in-vitro diagnosis and/or risk stratification of prostatic cancer.

As mentioned above, the tumor exosome nano fluorescence detection kit and the application thereof have the following beneficial effects: the invention not only proves that PSMA on the exosome derived from human plasma can be used as a biomarker for identifying healthy subjects and prostate cancer patients, but also further proves that the method can effectively avoid the influence of the exosome derived from normal cells in a clinical plasma sample on the detection result, has good anti-interference capability and has great clinical application potential in the aspect of prostate cancer PSMA (+) exosome subgroup analysis.

Drawings

FIG. 1 is a schematic diagram of a "Turn-on" type exosome nano-fluorescence sensing technology based on aptamer in the embodiment of the present invention.

Fig. 2 shows a map of the secretion profile of LNCaP cells from which the present invention was derived, wherein (a) is a transmission electron microscopy image, (b) is an NTA profile, and (c) is an immunoblot profile.

Fig. 3 shows a graph representing CD63 and PSMA in exosomes isolated from LNCap cell culture medium using two-channel super-resolution imaging technique in an embodiment of the present invention. (a) CD63 infects the exosome membrane protein CD 63; (b) alexa Fluor 647 fluorescently labeled PSMA; (c) and (3) carrying out double-channel super-resolution fusion on the image.

FIG. 4 is a diagram showing the detection results of aptamer-based "Turn-on" exosome nano-fluorescence sensing technology in the example of the present invention, wherein (a) P1+ TPE-TA; (b) exosome + P1+ TPE-TA + GO; (c) p1+ TPE-TA + GO; (d) fluorescence spectrum of TPE-TA.

FIG. 5 shows four PSMA aptamers in an embodiment of the invention: secondary structure and physical parameter diagrams of P1(a), P2(b), P3(c) and P4 (d).

FIG. 6 shows a statistical plot of fluorescence signal response values and a PAGE electrophoresis plot of an example of the invention, wherein (a) four PSMA aptamer sequences are compared for optimal aptamer selection; (b) PAGE shows that PSMA aptamer P1 can bind with prostate cancer LNCaP exosome, and lanes (1) to (6) are (1) DNA ladder (20-500bp), respectively; (2) p1; (3) p1+10⁷particles/μL exosome；(4)P1+10⁸particles/μL exosome；(5)P1+10⁹particles/μL exosome；(6)10⁹particles/μL exosome。

FIG. 7 is a graph showing the effect of TPE-TA concentration, GO concentration, reaction temperature and reaction time on fluorescence response in examples of the invention. (a) The TPE-TA reaction concentration is 1.6nM,3.2nM,4.8nM and 6.4nM fluorescence signal response value; (b) the reaction concentration of GO is 16 mug/mL, 24 mug/mL, 32 mug/mL and the response value of the fluorescence signal under the condition of 40 mug/mL; (c) the reaction temperature is 25 ℃,37 ℃ and 42 ℃ and the signal to noise ratio of the fluorescence response value is high; (d) the reaction time is 10min,15min,20min,30min,45min,60min and 75 min.

FIG. 8 is a diagram showing the detection specificity of aptamer-based "Turn-on" type prostate cancer exosome nano fluorescence sensing technology in the embodiment of the present invention.

FIG. 9 is a graph showing the sensitivity and linear detection range analysis in an embodiment of the present invention, wherein (a) the sensitivity and linear detection range analysis is based on aptamer-based "Turn-on" type prostate cancer exosome nano-fluorescence sensing technology. (a) Detecting fluorescence spectrograms acquired by PSMA (+) exosomes with different concentrations, wherein the concentrations of the exosomes corresponding to curves with different colors are sequentially increased from bottom to top along a vertical coordinate; (b) standard curves for different concentrations of PSMA (+) exosomes were tested.

FIG. 10 is a diagram showing the detection result of a clinical specimen by using aptamer-based "Turn-on" type prostate cancer exosome nano fluorescence sensing technology in the embodiment of the present invention. (a) Fluorescence response values were measured for plasma samples PSMA (+) exosomes of 7 healthy persons and 20 prostate cancer patients; (b) the scatter plot showed significant differences in PSMA (+) exosome concentrations between the healthy and prostate cancer patient groups.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The invention aims to specifically identify a Prostate cancer PSMA (+) exosome subgroup by using a Prostate Specific Membrane Antigen (PSMA) aptamer (aptamer), and an aggregation-induced luminescent molecule TPE-TA fluorescence reporter group and a Graphene Oxide (GO) fluorescence quenching group together form a label-free 'Turn-on' type fluorescence reporter system, so as to construct a Prostate cancer PSMA (+) exosome subgroup nano fluorescence biosensing detection technology. On the basis, analysis conditions are optimized, clinical diagnosis performance is verified, and the method aims to provide experimental basis and theoretical basis for constructing a new rapid and simple prostate cancer liquid biopsy method.

In order to construct a method for rapidly detecting exosomes with sensitivity and specificity and simple and convenient operation, the subject group closely focuses on the research progress of two essential elements required by the construction of an exosome nano fluorescence biosensing technology.

(I) identification element

The exosome membrane structure mainly comprises basic components such as phospholipid bilayers, membrane proteins, skeleton proteins, chaperonin and the like, and specific membrane protein components can mediate special functions of exosomes derived from different cells and also become main targets for detecting the exosomes derived from different cells. The latest research result proves that in addition to Membrane proteins such as transmembrane proteins (such as CD63, CD9 and CD81) and MHC class II molecules which are highly expressed by exosomes from different sources, in blood circulation of patients with early Prostate cancer, exosome surface specificity highly expressed Membrane protein Prostate Specific Membrane Antigen (PSMA) is adopted, so that the exosome subgroup with the PSMA Membrane protein specificity highly expressed, which is called PSMA (+) exosome subgroup for short, can be used as a target for screening and diagnosing early Prostate cancer to be applied to clinical detection.

The traditional membrane protein recognition element is mainly a membrane protein specific antibody, and in recent years, with the gradual and deep research on the in vitro screening technology, namely Exponential Enrichment Of Ligands By Exponential Enrichment Of SELEX, some aptamers (aptamers) capable Of specifically binding with membrane proteins are screened. Compared with antibodies, the aptamer has the advantages of stable chemical property, low immunogenicity, easy flexible modification by various groups and the like, and is widely applied to the step of target identification in the biosensing technology. For example, the CD63aptamer is coated on a gold electrode by Zhou, Q, and the like to construct an electrochemical biosensor system for quantitatively detecting exosomes in serum, the detection sensitivity of the kit is 100 times that of an anti-CD 63 antibody exosome detection kit, and the detection linear relation is stable. Therefore, this subject group was designed to screen PSMA aptamers for clinical use as an ideal recognition element for prostate cancer exosomes.

(II) fluorescent signal system

Because all cells of an organism can secrete exosomes, exosomes from specific tumor tissues or cells in peripheral blood circulation can be diluted by a large number of exosomes from normal cells, a fluorescent reporter group with high labeling efficiency, strong fluorescence intensity and low background is needed for sensitively indicating the specificity identification of the PSMA aptamer to the PSMA (+) exosome subgroup, and thus the quantitative detection of the PSMA (+) exosome subgroup in a clinical specimen is realized. At present, the nano fluorescence biosensing technology only depends on the traditional fluorescent dye and nano materials (such as quantum dots, up-conversion dots, nano particles encapsulating organic fluorescent molecules and the like) to mark biological substances, the materials may have the defects of low marking efficiency, low fluorescence intensity, high background, easy photobleaching, aggregated fluorescence quenching phenomenon at high concentration and incapability of meeting the quantitative detection requirement of low-abundance exosomes.

Aggregation-Induced Emission (AIE) molecules are a new type of nano fluorescent molecules with high fluorescence signal-to-noise ratio and capable of overcoming the Aggregation fluorescence quenching defect of the traditional fluorescent substance, and have attracted extensive attention in the fields of chemistry, material science, life science and the like so far. Particularly in the technical field of biosensing, because the AIE nano-molecule has good solubility and biocompatibility and has the characteristics of aggregation, induction and luminescence, the AIE nano-molecule can be used for constructing a 'Turn-on' type fluorescence sensing technology suitable for a liquid phase detection system, the technology has simple and convenient design and strong practicability, and is particularly suitable for developing a novel method for high-sensitivity tumor exosome biopsy with simple and convenient operation. Hong Y et al report that an AIE nano-molecule TTAPE (tetraphenylethene derivitives) specifically binding to DNA/RNA molecules has substantially no fluorescence emission in a free state, and when DNA/RNA molecules are present in a solution, a large amount of TTAPE is aggregated on a DNA/RNA strand and initiates aggregation-induced luminescence effect, and a fluorescence emission peak appears at 480 nm. Whereas aptamers are essentially single-stranded dna (ssdna) molecules, TTAPE can quantitatively detect PSMA (+) exosome subpopulations by aggregation-induced emission effects on PSMA aptamers.

However, due to the presence of free aptamers that do not bind to exosomes in the detection system, these aptamers can also aggregate TTAPE and fluoresce, thereby interfering with the detection signal of the PSMA (+) exosome subpopulation. Graphene Oxide (GO) is a derivative of Graphene, contains various oxygen-containing functional groups, and exhibits excellent hydrophilicity and processability. Besides the high oxidation region, a graphite-like region which is not oxidized exists on the surface, and the graphite-like region enables graphene oxide to maintain strong adsorption capacity to biological molecules and has effective adsorption and fluorescence quenching effects on organic fluorescent molecules. Studies have proved that GO can effectively adsorb ssDNA molecules and AIE nano molecules. Therefore, in order to improve the detection specificity of a fluorescent signal system, the subject group intends to establish a 'Turn-on' type fluorescent report system by adopting nano molecules TTAPE and GO, and when no prostate cancer PSMA (+) exosome subgroup exists in the detection system, the free PSMA aptamer is adsorbed by GO and quenches TTAPE fluorescent signals gathered on the free PSMA (+) exosome subgroup; when a prostate cancer PSMA (+) exosome subgroup exists, PSMAAptamer is combined with a target exosome, falls off from the surface of GO and enables TTAPE gathered on GO to emit light, a Turn-on type fluorescence detection signal appears, and the strength of the signal is in positive correlation with the concentration of the PSMA (+) exosome subgroup in a solution.

Based on previous research results and referring to a large amount of literature data, the subject is supposed to specifically identify the prostate cancer PSMA (+) exosome subgroup by utilizing PSMAaptamer, and an AIE molecule TTAPE fluorescence reporter group and a GO fluorescence quenching group form a label-free 'Turn-on' type fluorescence reporter system together, so that the prostate cancer PSMA (+) exosome subgroup nano fluorescence biosensing detection technology is constructed. On the basis, analysis conditions are optimized, clinical diagnosis performance is verified, and the method aims to provide experimental basis and theoretical basis for constructing a new rapid and simple prostate cancer liquid biopsy method.

The PSMAaptamer is used for specifically identifying the PSMA (+) exosome subgroup of the prostate cancer, an AIE molecule TPE-TA fluorescence reporter group and a GO fluorescence quenching group are combined to form a label-free 'Turn-on' type fluorescence reporter system for the first time, and a nano fluorescence biosensing detection technology for the PSMA (+) exosome subgroup of the prostate cancer is established by the label-free 'Turn-on' type fluorescence reporter group, so that the quantitative detection of the PSMA (+) exosome subgroup of the prostate cancer is realized. The experimental results of the part prove that the specificity of PSMAAptamer for identifying the prostate cancer exosomes and the feasibility of a 'Turn-on' type fluorescence report system based on TPE-TA and GO, after prostate cancer LNCaP cell source exosomes with certain concentration are added, the fluorescence response value of the nano fluorescence biosensing detection technology is obviously increased, and compared with a control group (c), the signal-to-noise ratio (S/N) of the fluorescence response value is about 6 times. Based on the above, important reaction conditions (mainly including TPE-TA concentration, GO concentration, reaction temperature and reaction time) involved in the experiment are researched, and according to the analysis of experimental results, when the optimal reaction concentration of the fluorescence reporter group TPE-TA is 3.2nM, the optimal reaction concentration of the fluorescence quenching group GO is 32 mug/mL, and under the mild reaction condition of 37 ℃, the fluorescence signal can reach the maximum value only through 15min of reaction time, and the reaction condition can be quickly completed on a portable thermostat, and the result can be interpreted in a short time, so that the simplicity and the high efficiency of the detection method are further proved.

Further, we performed a preliminary methodological evaluation of the quantitative detection method for this prostate cancer PSMA (+) exosome subpopulation. First, we evaluated the specificity of this nano fluorescence sensing technology for the detection of prostate cancer cell-derived PSMA (+) exosome subpopulation. The result shows that the exosome subgroup detection method established based on the nano fluorescence sensing technology can effectively distinguish the prostate cancer LNCaP cell source PSMA (+) exosome subgroup from other tumor cell lines or normal cell source exosomes, can avoid the interference of the normal cell source exosomes in plasma on the detection result, and has good specificity. Secondly, we use this method under the optimum reaction conditionsA series of prostate cancer LNCaP cell-derived exosomes with different concentrations are detected, and the fluorescence intensity at 480nm of the strongest emitted light is analyzed to evaluate the linear range and sensitivity of the method for detecting the prostate cancer cell-derived PSMA (+) exosome subpopulation. The result shows that the fluorescence signal of the method is in positive correlation with the concentration of the exosome from the prostate cancer LNCaP cell source, and the linear detection range of the fluorescence signal is 4.07 multiplied by 10⁵-1.83×10⁷mu.L, the lowest detection limit is 3.43 multiplied by 10⁵Compared with other exosome rapid detection technologies established based on nucleic acid aptamers, the oligonucleotides/mu L nucleic acid aptamer has the advantages of simpler operation, shorter reaction time, similar sensitivity and obvious application advantages.

In addition, in order to further verify the clinical application potential of the constructed nano fluorescence sensing technology, 20 cases of prostate cancer patients and 7 cases of healthy human plasma specimens are collected clinically, and under the optimal reaction condition, the method provided by the invention is used for carrying out fluorescence detection on plasma source exosomes of a prostate cancer patient group and a healthy human group so as to evaluate the clinical detection performance of the method. The result shows that the concentration of PSMA (+) exosomes of a prostate cancer patient is obviously higher than that of healthy people, and the experiment not only proves that PSMA on exosomes in human blood can be used as a biomarker for identifying healthy subjects and prostate cancer patients, but also shows that the method can effectively avoid the influence of normal cell source exosomes in clinical plasma samples on the detection result, and has clinical application potential in the aspect of prostate cancer PSMA (+) exosome subgroup analysis.

1. Principle explanation

In the invention, ssDNAPAMAaptamer is used as a PSMA (+) exosome recognition element, AIE molecule TPE-TA is used as a fluorescence reporter group, and GO is used as a fluorescence quenching group to jointly construct a label-free 'Turn-on' type fluorescence reporter system so as to realize the quantitative detection of the PSMA (+) exosome. The detection principle is shown in figure 1, after ssDNAPAMABetamer reacts with TPE-TA, the TPE-TA is gathered on a PSMAPAMABetamer single-stranded nucleic acid sequence to form an aptamer/TPE-TA compound, and the fluorescence intensity in the solution is rapidly enhanced due to aggregation-induced luminescence effect. And then, adding GO into the reaction solution, wherein due to the high-strength binding force between the aptamer and GO, the aptamer/TPE-TA compound is adsorbed on the surface of GO, and the fluorescence signal of the aptamer/TPE-TA is effectively quenched under the action of fluorescence energy resonance transfer between AIE molecules and GO. When PSMA (+) exosomes exist in the solution, aptamer/TPE-TA compounds can be effectively combined with the PSMA (+) exosomes, so that the combination between the aptamer/TPE-TA compounds and GO is weakened, the fluorescence quenching effect of GO on AIE molecules is further reduced, and the fluorescence of the aptamer/TPE-TA compounds combined on the surfaces of the PSMA (+) exosomes can be gradually recovered.

2. Materials and methods

2.1 materials

2.1.1 cell lines

Human prostate cancer cell lines: LNCaP purchased from cell resource center of Shanghai Life sciences research institute of Chinese academy of sciences and stored in liquid nitrogen.

2.1.2 Primary reagent consumables

TABLE 1 summary of reagent consumables

References to TPE-TA molecules are: Dual-Mode Ultrasensitive Detection of Nucleic Acids via an Aqueous "Seesaw" Stratagene by Combining Aggregation-Induced emission and plasma Colorimetry, Jianlei Shen, Yiru Zhang, Rong Hu, Ryan T.K.Kwok, Zhiming Wang, Anjun Qin, and Ben Zhong Tang, ACS appl.Nano Mater.2019,2, 163-169.

The specific structure of TPE-TA molecule can also refer to the Chinese patent of invention publication No. CN 108949919A, a aggregation-induced emission/surface plasma colorimetric analysis dual-mode nucleic acid detection method, the description of which [ 0038-; xiong, h.; lam, J.W.Y.; ha u β ler, m.; liu, j.; yu, y.; zhong, y.; sung, h.h.y.; williams, i.d.; wong, k.s.; tang, B.Z.Fluorogenic Bioprobes Structural Matching in the packaging process of Aggregation-Induced Emission Fluorogens on DNAsurfaces chem. -Eur.J.2010,16,1232-1245 ".

2.1.3 Main Instrument

TABLE 2 Instrument name and manufacturer summary sheet

TABLE 3 nucleic acid sequence Listing as referred to in this example

2.2 methods

2.2.1 isolation and purification of exosomes from cell supernatants of prostate cancer LNCaP cell cultures

(1) Cell culture

1) Cell recovery: firstly, taking out the freezing tube of the prostate cancer LNCaP cell strain frozen in a liquid nitrogen tank, quickly transferring the freezing tube into a thermostatic waterbath box at 37 ℃ to quickly dissolve the prostate cancer LNCaP cell strain, and shaking the freezing tube from time to time during the incubation process to uniformly heat the freezing tube. After the cells are completely dissolved, adding 5mL of RPMI-1640 culture solution into a 15mL centrifuge tube, blowing and uniformly mixing the cell suspension in the frozen tube, transferring the cell suspension into the centrifuge tube, centrifuging the cell suspension at 1200rpm for 2min, discarding the supernatant in the centrifuge tube, then adding 5mL of complete culture medium (10% fetal calf serum and 90% RPMI-1640 culture solution), re-suspending and uniformly mixing the cell suspension, transferring the cell suspension to 25cm²Placing in a cell culture flask at 37 deg.C and 5% CO₂After culturing in the humid constant temperature incubator for 2-3 days, observing the state and density of cells, and waiting for the cells to grow and propagateSubculture until 70-80% of the bottom area of the covered culture flask or culture dish.

2) Cell passage: adopting a pipette gun to discard old culture medium in a culture bottle or a dish, washing the culture bottle or the dish for 2-3 times by PBS, adding 1mL of pancreatin, slightly shaking the culture bottle or the dish to uniformly distribute the pancreatin on the cell surface, digesting cells by the pancreatin for about 1min, observing under an inverted microscope, adding 200 muL of serum to stop digestion if the cell gap is obviously increased and the cell morphology begins to become round, adding a proper amount of PBS to blow and beat the cells gently, transferring the cells to a 15mL centrifuge tube after the cells are separated from the bottom of the culture bottle or the dish and are uniformly mixed, centrifuging at 1200rpm for 3min, adding RPMI-1640 culture solution containing 10% fetal bovine serum after supernatant is sucked and discarded, and resuspending the culture solution 1: 3 or 1: 4 passages, then placing at 37 ℃ and 5% CO₂The wet constant temperature incubator.

3) Collecting and pretreating cell supernatant: observing the growth condition of LNCaP cells, discarding the old culture medium in the bottle or dish when the cells grow and propagate to cover 60-70% of the bottom area of the culture bottle or dish, washing the bottle or dish with PBS 2-3 times, adding serum-free RPMI-1640 culture solution, then culturing in the incubator for 12h (removing the influence of secretion exosomes of residual serum) under the same condition, then discarding the old culture medium in the bottle or dish, washing the bottle or dish with PBS 2-3 times, adding 1-2% Exo-FBS^TMAnd (removing the exosome fetal calf serum) continuously culturing the RPMI-1640 culture solution for 48 hours to ensure that the cells secrete a certain amount of exosomes into the culture solution. Collecting LNCaP cell supernatant after 48h for pretreatment, and specifically comprising the following steps: centrifuging at 300g for 10min to remove residual cells in the culture supernatant, discarding the precipitate, and collecting the supernatant; centrifuging at 2,000g for 20min to remove cell debris from the supernatant, discarding the precipitate, and collecting the supernatant; centrifuging at 10,000g for 30min, removing larger vesicles from the supernatant, discarding the precipitate, and collecting the supernatant.

(2) Separation and purification of exosomes in cell supernatant

Centrifuging for 70min by using a low-temperature ultrahigh-speed centrifuge L-80XP135,000g to obtain a precipitate as an exosome, adding a proper amount of PBS to re-suspend and uniformly mix, then continuing centrifuging for 70min by using 135,000g to remove impurity proteins dissolved in the PBS, finally obtaining the precipitate as a relatively pure exosome, finally re-suspending and precipitating by using 100 mu L of PBS, namely the separated and purified exosome, and storing in a refrigerator at the temperature of-80 ℃ for later use.

2.2.2 Transmission Electron microscopy of morphological Structure of prostate cancer LNCaP cell-derived exosome

Diluting the separated and purified exosome by different times, sucking 15 mu L of exosome sample from the exosome sample, dripping the exosome sample on a copper mesh special for an electron microscope, standing for 2min at room temperature, sucking the liquid from the other side of the copper mesh by using filter paper, then dripping 20 mu L of 2% phosphotungstic acid staining solution on the copper mesh, standing for 10min at room temperature, sucking the redundant phosphotungstic acid, washing for 1-2 times by using distilled water, sucking the distilled water by using the filter paper, standing for 10min at room temperature, and drying the copper mesh. The copper mesh loaded with the sample is clamped by a pointed-end tweezers and placed in the sample mounting groove, the gasket is mounted and fixed, then the sample tube is aligned to the sample inlet of the transmission electron microscope, the sample tube is pushed into the sample mounting groove by a thumb slightly at the bayonet, and the red light is lightened after the sound is heard. Turning on a switch at the lower part, turning on a green lamp to light, rotating the sampling tube rightwards, automatically sucking the sample, then rotating the sampling tube around 5 ℃ to suck the sample into the bottom, and observing the form of the exosome under a transmission electron microscope H-7650 and taking a picture.

2.2.3 NanoSigt NS300 analysis of prostate cancer LNCaP cell-derived exosome concentration and particle size distribution

The ideal upper computer particle concentration for the NanoSight NS300 analyzer is 1 × 10⁸-1×10⁹particles/mL, therefore, the isolated and purified exosomes need to be diluted before loading to make the concentration in the ideal detection range, and the loading sample amount is 1 mL. Before analyzing the sample, PBS is used as a blank control, a 405nm laser is selected, the background value of the detection chamber is observed, and the PBS is used for cleaning the pipeline and the detection chamber until no particle bright spots appear in the background. Then 1mL of the diluted sample solution was aspirated with a 1mL syringe, slowly pushed into the tube, and after the flow stabilized, video shots were taken, each for 60s (30frames/s) of video. In order to ensure accurate results, sample introduction and detection are generally repeated three times, and the stability of the operation table surface is kept during shooting. Then, a threshold is set for the particles in the captured video, and the counts of ion false positives and false negatives are reduced. The brownian-moving particles in the video were then subjected to a process based on ntasoft ware (Version 3.2, NanoSight) softwareAnd calculating and analyzing, namely combining the scattering intensity of the particles with an Einstein equation to obtain the concentration and the hydrodynamic diameter of the detected particles, and drawing a three-dimensional map of the scattering intensity, the concentration and the particle size distribution intensity of the particles. And finally, pumping the sample in the detection chamber back to the injector, wiping the residual sample on the laser by using a piece of mirror wiping paper after the laser and the detection channel part are disassembled, cleaning the detection channel by using water, airing and placing for later use.

2.2.4Western Blot to verify the content of CD63 protein and PSMA protein on exosomes derived from prostate cancer LNCaP cells

(1) Preparation of the principal solution

10% Ammonium Persulfate (AP): 0.1g of ammonium persulfate is dissolved by adding 1mL of deionized water, and the dissolved ammonium persulfate is stored in a refrigerator at 4 ℃.

10% SDS: adding deionized water into SDS powder according to the weight (g) of SDS/final volume (mL) of the solution being 1:10 for constant volume, mixing uniformly, filtering by using filter paper, and storing at room temperature.

5 XTris-glycine electrophoresis buffer solution

And (4) storing at room temperature after dissolving, and repeatedly using the solution for 3-5 times.

1 Xtransfer buffer

And (4) storing at room temperature after dissolving, and repeatedly using the solution for 3-5 times.

TBST buffer

Blocking solution (TBST buffer containing 5% skimmed milk powder): TBST was added to 1g of skim milk powder to a constant volume of 20mL, and the mixture was stored at room temperature.

10% separation gel and 5% concentration gel, and TEMED is added before filling.

TABLE 4 Table of the components of the separation gel and the concentrated gel

(2) Exosome protein extraction and quantification

1) Extraction of exosome proteins

Preparing SDT lysate with pH of 7.6 from 4% SDS, 100mM Tris-HCl and 1mM DTT, adding 100 mu L lysate into the exosome suspension, fully lysing, centrifuging for 15min at 14000g, and taking supernatant as exosome protein extract.

2) Quantification of exosome protein concentration

And (3) determining the protein concentration of the sample by using a ThermoFisher micro BCA protein quantitative kit. Preparing protein solution and sample to be detected with corresponding concentration by using protein standard solution in the kit, respectively sucking 25 μ L of sample into 96-well plate, respectively adding 200 μ L of working solution L (by volume, A: B ═ 1: 50; A: 1% BCA, 2% Na)₂CO₃·H₂O，0.16％Na₂C₄H₄O₆·2H₂O，0.4％NaOH，0.95％NaHCO₃pH: 11.25; and B, liquid B: 4% CuSO₄·5H₂And O. ) Shaking for 30s, thoroughly mixing, sealing, incubating in 37 deg.C water bath for 30min, cooling to room temperature, and measuring absorbance of sample with microplate reader at 562nm wavelength. And drawing a standard curve by taking the protein content (mu g) as an abscissa and the light absorption value as an ordinate. And (3) searching the corresponding protein content (mu g) of the sample to be detected on a standard curve according to the detected absorbance value, and converting the protein concentration and the sample loading amount according to the volume of the suspension.

3) Protein sample denaturation: adding a Loading buffer into the protein extract according to the volume ratio of the protein sample to the Loading buffer of 4:1, boiling water bath for 10min, and then storing in a refrigerator at-20 ℃.

(3) SDS-PAGE electrophoresis

1) Stacking two cleaned glass plates, aligning the bottoms of the two cleaned glass plates, fixing the two glass plates on a bracket, adding double distilled water to the upper edges of the glass plates, waiting for 20-30min, evaluating the liquid level descending condition, re-fixing the two glass plates if the liquid level descending condition is more than 5mm, pouring the double distilled water after sealing, and sucking the double distilled water by using filter paper (a small amount of residual water does not influence glue filling).

2) Glue filling

Preparing 10% separation gel according to a formula, finally adding AP and TEMED, mixing uniformly after adding TEMED, and immediately pouring gel; adding separation glue to 2/3 wide part of the small glass plate, about 1cm below the lower edge of the comb (optionally adding more glue according to glue leakage), immediately adding double distilled water to the edge of the small glass plate, standing for 30min (shortened to 20min in summer), observing obvious boundary line, indicating that the separation glue has solidified, pouring out the double distilled water on the glue, and sucking the residual water with filter paper; adding 5% concentrated glue to the upper edge of the small glass plate, immediately inserting the sample comb, inserting the comb in parallel while avoiding generation of bubbles, standing at room temperature for 30-45min to solidify.

3) Sample loading

Carefully pulling out the comb after gelation, taking the rubber plate off the rubber frame, oppositely fixing the two small plates in an electrophoresis tank, adding SDS electrophoresis buffer solution between the two glass plates until the upper edge of the small plate is 5mm, and pouring a proper electrophoresis buffer solution between the electrophoresis tank and the glass plates; and (3) using a micro syringe to sample, sucking 5 mu L of the pre-dyed protein Marker and 10 mu g of the sample, extending the needle tip to the bottom of the sample adding hole during sample adding, and slowly and carefully adding.

4) Electrophoresis

And (3) concentrating the sample into a narrow band by using the low voltage of 80V for 20min in the gel concentration part, and selecting the high voltage and constant voltage current of 120V when the band moves to the separation gel until the bromophenol blue reaches the bottom to stop electrophoresis.

(4) Rotary film

Cutting the glue: and taking off the rubber plate, carefully separating the large glass plate and the small glass plate to avoid the glue from being damaged by traction and prevent the glue from drying, cutting off a required part according to the molecular weight shown by a Marker and the molecular weight of the target protein, and cutting out 6 pieces of filter paper and a PVDF film (the cut corners are used for marking the front side and the back side) with the same size according to the size of the cut glue. Soaking the PVDF membrane in 100% methanol for about 10s, then adding distilled water to rinse for 2-3min, then transferring to a membrane conversion buffer solution to balance for 5min, and placing the filter paper in the membrane conversion buffer solution to soak for 3-5 min.

Preparing a rotary film clamp (sandwich): the rotating film clamp is opened to enable the black side to be kept horizontal, a spongy cushion is arranged on the rotating film clamp, bubbles are removed through a glass rod, 4 layers of filter paper are arranged on the sponge cushion, the filter paper is fixed by one hand, and the bubbles are removed through the glass rod by the other hand. The cut glue was placed flat on filter paper, the film was covered over the glue, the whole glue was covered (the film was not allowed to move after covering) and the air bubbles were removed. The membrane was covered with 4 sheets of filter paper and the air bubbles were removed. Covering the sponge cushion on the other side, removing bubbles in the sponge cushion by using a glass rod, and closing the clamp. The whole operation is carried out in the transfer liquid, and air bubbles are continuously rolled out. Turning on the film transferring clamp, wherein black is opposite to black, white is opposite to red, putting the film transferring clamp into a film transferring groove, fully adding a film transferring buffer solution into the film transferring groove, putting into an ice bag, and oppositely connecting the same-color electrodes with a power supply; the membrane-rotating tank was placed in cold water and ice bags, and 200A was galvanostatrically transferred for about 1h (Bio-rad wet rotation).

(5) Sealing of

And opening the membrane transferring clamp, taking out the membrane, and indirectly proving that the protein is successfully electrotransferred when the marker is transferred on the PVDF membrane. The membrane is taken out and placed in a sealing solution, the shaking table is sealed for 2 hours at the room temperature at the speed of 80rpm/min, and for the test with poor antibody specificity, the membrane can be placed in a refrigerator at the temperature of 4 ℃ for incubation overnight so as to seal the non-specific sites on the membrane.

(6) Primary antibody and secondary antibody incubation

Respectively adding primary anti-dilution solution with different proportions of CD63 and PSMA, sealing with plastic film, and incubating overnight at 4 deg.C on a refrigerator shaker; washing the membrane with TBST for 5min for 3 times the next day; respectively adding diluted horseradish peroxidase labeled anti-rabbit secondary antibody diluent, incubating for 1-2h in a shaking table at room temperature, and washing the membrane for 3 times by TBST, each time for 10 min.

(7) Chemiluminescence development

Mixing A, B solutions according to a volume ratio of 1:1 (about 1 mL), placing in a plate (solution A is hydrogen peroxide solution, solution B is luminol solution, and solution A and solution B are purchased from Frade bioscience, Hangzhou, and the product information is FD Fdbio science, size:100mL, Fdbio-Dura ECL Kit, Enhanced cheminescence Kit HRP, Store at 4 ℃, Cat No: FD8020, Lot No: 20190120); gently draining the membrane on filter paper, and adding into the uniformly mixed A, B liquid; the bands were scanned using a ChemiDocXRS imaging system and the protein expression levels shown for each band were analyzed.

2.2.5 super-resolution microscopy imaging verification of expression of CD63 antibody and PSMA on exosome membranes

(1) Exosome super-resolution imaging operation steps

1) PLL-coated coverslip, which was coated with 50. mu.L of 1mg/mL Poly-L-lysine on a petri dish, incubated at room temperature for 30min, and washed 3 times with an appropriate amount of PBS.

2) mu.L of the sample + 40. mu.L of PBS (set at different concentrations), 50. mu.L were placed in a petri dish and incubated at room temperature for 30 min.

3) Add 50. mu.L Dilution C and 0.25. mu.L PKH67 to the shaded EP tube and mix well.

4) The mixed suspension of PKH67 was added to a petri dish, incubated at room temperature for 4min, and shaken gently.

5) After the solution was aspirated, 100. mu.L of 1% BSA was added and incubated for 2min, and washed three times with an appropriate amount of PBS.

6) The purchased CD63 antibody was expressed as 1: 400 volume ratio dilution, 50 u L diluted CD63 antibody incubated for 1h, appropriate PBS washing three times.

7) The following compositions were used: goat anti-rabbit Alexa Fluor 647 labeled fluorescent secondary antibody at 2000 dilutions (volumes) was incubated for 40min and washed 3 times with PBS.

8) PBS was applied to cover the sample area and stored at 4 ℃.

9) The PBS was aspirated and replaced with super-resolution imaging buffer to cover the entire sample area.

10) Images were captured after irradiation with 488nm and 647nm wavelength lasers using a nikon N-SIM super resolution microscope system.

2.2.6 aptamer-based feasibility verification experiment of 'Turn-on' type exosome nano fluorescence sensing technology

Synthesis of a PSMA aptamer based on the literature reported PSMA aptamer P4 nucleic acid sequence (see Table 3) (reference)The literature: development of a Single Stranded DNA Aptamer a Molecular Probe for LNCap Cell Using Cell-SELEX, Faezeh Almasi, Seye Latif Mousavi Gargari, Fatemeh Bitaraf, and Samaneh Rasouleinejad, Avicenna Journal of Medical Biotechnology, Vol.8, No.3, July-September 2016), 60 μ L of 3.2nM TPE-TA neat solution, 60 μ L of 3.2nM TPE-TA solution containing 1 μ M PSMA Aptamer and 32 μ g/mL GO, and 60 μ L of 3.2nM TPE-TA solution and 1 × 10 μ L TPE solution with concentration⁸Carrying out fluorescence signal detection on a PSMA aptamer/GO/TPE-TA solution subjected to co-incubation of prostate cancer LNCaP cell source exosomes/mu L on a fluorescence spectrophotometer, and verifying that after the PSMA aptamer identifies the prostate cancer LNCaP cell source PSMA (+) exosome subgroup, the aggregation-induced luminescence phenomenon is generated by a 'Turn-on' type fluorescence report system based on TPE-TA and GO.

2.2.7 fluorescent Signal response detection

After the fluorescence spectrophotometer and software are turned on, "Status" is selected in the "Application" menu and the instrument Status (including light source, light path system, sample cell and signal detection) is observed. And selecting 'Validation' under the condition that the instrument is in a correct state, and confirming the state of the instrument by filling a matched dish with deionized water. The detection dish is cleaned by 95% alcohol, and then is repeatedly cleaned by deionized water for three times and then is dried by nitrogen. Setting the fluorescent signal response value detection condition: the scanning wavelength range is 370nm-650nm, the exciting light is 350nm, the slit of exciting light and emitting light is 10nm, the scanning speed is 700nm/min, and the fluorescence intensity of the strongest emitting light at 480nm is analyzed. Under the condition, 60 mu L of deionized water is detected, and when the fluorescence intensity in the scanning range is less than 1, the specimen can be detected; otherwise, the washing is continued until the detection dish is qualified.

2.2.8 screening and validation of PSMA (+) exosome subgroup-specific PSMA aptamers

(1) PSMAAptamer screening

The method searches a PSMAAptamer related document in a PubMed database, and finds out four reported PSMA aptamers, namely P1, P2, P3 and P4. The DNA sequences of the four PSMA aptamers were verified in the aptamer database Aptagen (see table 3) and the secondary structures of the four aptamers were predicted using the online software upnp ack.

Four DNA sequences of PSMA aptamers were synthesized using 1. mu.M of four PSMA aptamers in 1X 10 solution (DEPC water as solvent)⁸And (3) incubating each/mu L of prostate cancer LNCaP cell-derived exosomes (dissolved by PBS buffer solution), adding 3.2nM TPE-TA molecules for dark reaction, adding 32 mu g/mL GO to adsorb free aptamer and TPE-TA molecules, finally performing fluorescence signal detection on a fluorescence spectrophotometer, and selecting the PSMA aptamer most suitable for the test according to the fluorescence signal-to-noise ratio (S/N).

TABLE 5 reaction reagent table

(2) Verification of the ability of the optimal PSMA aptamer to recognize a subpopulation of PSMA (+) exosomes by non-denaturing polyacrylamide gel electrophoresis

1) Repeatedly cleaning a required thick glass plate (1mm edge strip), a required short glass plate, a glue making comb, a glue making bracket and a glue making frame, then washing the glass plate by deionized water, and airing the required tools.

2) Insert the recess of vertically system frame after aligning clean glass board, notice the arrow point up, the both sides are pressed from both sides tightly fixedly with the clip, and the bottom is adjusted and is ensured the lower extreme parallel and level to fix system frame on system gluey support.

3) And filling deionized water into a gap between the thick glass plate and the short glass plate, observing for 10min, detecting leakage, and preparing the glue after ensuring that a glue preparation system does not leak liquid.

4) Pouring out the water in the glue making frame by tilting, and fully absorbing residual water at each corner of the glue making frame by using filter paper for later use.

5)5 × TBE buffer: 54g of Tris-base, 27.5g of boric acid and 4.65g of EDTA are weighed by an electronic analytical balance, 500mL of deionized water is added, a glass rod is used for stirring until the solution is completely dissolved, the volume is determined to be 1L, and the mixture is stored at normal temperature.

The procedure for preparing 0.5 × TBE buffer was as follows: the cylinder accurately measures 50mL of 5 XTBE buffer, and adds 450mL deionized water to dilute, and fully shake before use.

6) 10% AP: weighing 1g of ammonium persulfate powder by using an electronic analytical balance, adding 5mL of deionized water for dissolution, stirring by using a glass rod until the solution is completely dissolved, then metering the volume to 10mL, and storing at 4 ℃.

7) NaCl solution (1M): 58.44g of NaCl powder is weighed by an electronic analytical balance, 500mL of deionized water is added, the mixture is stirred by a glass rod until the solution is completely dissolved, then the volume is determined to be 1L, and the mixture is stored at normal temperature.

8)3 × nucleic acid staining solution: 45mL of deionized water and 5mL of 1M NaCl solution were added to 15. mu.L of 4S Red Plus nucleic acid stain (10000X), mixed well and stored at 4 ℃ in the dark. The nucleic acid staining solution is required to be balanced to room temperature before use, and can be recovered and reused for 3 times.

9) Preparation of the gel

According to the molecular weight of the PSMA aptamer, 12% of glue is selected, and the specific components are shown in a table 1.4:

TABLE 6 preparation of PAGE gels formula Table (12% Gel)

10) Glue injection

And (3) after the gel solution in the step 9) is prepared according to the volume, fully and uniformly mixing, quickly and slowly filling the prepared gel solution along the gap between the thick glass plate and the short glass plate by using a sample adding gun, and avoiding the generation of bubbles in the adding process. Then, a thick glass rod is attached, the glue making comb is gently inserted into the gel solution between the gaps of the two glass plates, and careful operation is carried out to avoid generating bubbles, and the bubbles need to be removed completely if necessary. And then placing the gel making frame in a water bath tank at 37 ℃ for 20-40 min until the solution is solidified. After the gel is completely solidified, the glue making comb is carefully pulled out vertically upwards, and the solidified glue is placed in 0.5 XTBE buffer along with the glass plate for storage.

11) Sample loading

According to the inward direction of the short glass plate, the two pieces of glue are carefully clamped on the electrode core along with the glass plate, then the electrode core is fixed on the electrophoresis tank according to the corresponding electrode, and the space between the two pieces of glue (the inner groove) is filled with 0.5 xTBE buffer solution, preferably being parallel and level to the upper edge of the thick glass plate. PSMA aptamer was mixed at different concentrations (1X 10) in a 5:1 ratio by volume⁶,1×10⁷,1×10⁸One/. mu.L) prostate cancer LNCaP exosomes were co-incubated at 37 ℃, the incubation product was mixed well with 6 × loading buffer, carefully added to the sample wells for a total volume of about 6. mu.L, and 5. mu.L of 20bp DNA ladder marker was added to each gel.

12) Electrophoresis

According to the number of the electrode cores, 0.5 times TBE buffer solution is poured into the outer tank of the electrophoresis tank to the position of the scale mark. Connecting electrodes, turning on power supply, regulating voltage to 150V, and electrophoresis time about 30min to indicate that the tape should be electrophoresed to the position of 2/3. After electrophoresis to the desired position, the power was turned off, the buffer was recovered, the glass plate was removed and uncovered, the gel carefully removed and rinsed clean with deionized water.

13) Nucleic acid staining

Pouring the diluted 3X 4S Red Plus nucleic acid staining agent into a clean container, putting the gel, covering the container with aluminum foil paper to prevent the staining agent from light, placing the container on a shaker, and uniformly mixing at 60rpm for about 30 min.

14) Image display

Placing the Gel in an ultraviolet Gel imaging system, opening a TRANS UVI, selecting a Gel Doc XR scanner, adopting an Auto Exposure mode, clicking a Freeze frozen image when a nucleic acid strip is clearly displayed, and storing and exporting the result.

2.2.9 Effect test of reaction conditions

And respectively researching the influence of TPE-TA concentration, GO concentration, reaction temperature and reaction time conditions on the response value of the fluorescent signal. Sequentially detecting the reaction concentrations of TPE-TA to be 1.6nM,3.2nM,4.8nM and 6.4nM respectively, the reaction concentrations of GO to be 16 mug/mL, 24 mug/mL, 32 mug/mL and 40 mug/mL respectively, the reaction temperatures to be 25 ℃,37 ℃ and 42 ℃ respectively, and the reaction time to be 10min,15min,20min,30min,45min,60min and 75min respectively, calculating the signal-to-noise ratio of the fluorescence response value based on the fluorescence signal response value of the nucleic acid aptamer 'Turn-on' type exosome nano fluorescence sensing detection method, and searching the optimal reaction condition of each parameter.

2.2.10 specificity assays

Collecting the supernatant of LNCaP cell culture of prostate cancer cell line, extracting exosome by ultracentrifugation, using the exosome extracted by the same method as breast cancer cell line MDA-MB-231, normal liver cell line HL7702, normal macrophage line RAW264.7 cell culture supernatant and normal human plasma source exosome as control group, observing the particle size distribution by using NanoSight, and unifying the concentration to 1 × 10⁸mu.L/L for detection. The nucleic acid aptamer-based Turn-on type exosome nano fluorescence sensing detection method is used for detecting the five exosome samples, the response value of each group of fluorescence signals is recorded, the signal-to-noise ratio of the fluorescence response value is calculated, each exosome sample is repeatedly detected for three times, and the detection specificity of the method is evaluated.

2.2.11 sensitivity analysis

Under the optimized optimal experimental conditions, 3 mu L of LNCaP cell source exosomes with different concentrations are respectively added into a nucleic acid aptamer-based Turn-on exosome nanometer fluorescence sensing detection system for reaction, a fluorescence signal response value is detected by a fluorescence spectrophotometer after the reaction is finished, and the signal-to-noise ratio of the fluorescence response value is calculated. And (3) repeatedly detecting the exosome sample at each concentration for three times, recording the detection result, and calculating and analyzing to obtain a linear range and a detection lower limit of the PSMA (+) exosome subgroup from the prostate cancer LNCaP cell source so as to explore the detection sensitivity of the method.

2.2.12 assessment of detectability of clinical specimens

Plasma specimens of 20 prostate cancer patients and 7 healthy persons were collected at southern hospital laboratory of southern medical university, 500. mu.L of the plasma specimens were centrifuged at 300g, 2,000g and 10,000g, centrifuged at 54,000rpm for 2 hours by a microcentrifuge, and the precipitated PBS was collected for resuspension and centrifuged under the same conditions to collect plasma-derived exosomes. The concentration of exosomes in plasma samples was measured using NTA, and subsequent experiments were performed with the same exosome concentration. Under the optimal reaction condition, the nano fluorescence sensing detection method is used for carrying out fluorescence detection on the plasma source exosomes of a prostate cancer patient group and a healthy human group, and the experiment is repeated for three times to evaluate the detection capability of the nucleic acid aptamer-based Turn-on type exosome nano fluorescence sensing technology clinical specimen.

1) Purification of exosomes in cell supernatant by TEM, NanoSight and Western Blot identification ultracentrifugation method

And purifying the exosome derived from the prostate cancer cell line LNCaP cell line by using an ultra-high speed centrifugation method. As shown in FIG. 2(a), TEM image showed that the exosomes extracted by ultracentrifugation had an intact structure of membrane lipid bilayer and diameter around 100nm, which is compared with literature report (Shao Huilin, Im Hyungsoon, Castro Cesar M et al.New Technologies for Analysis of excellular vehicles [ J.]Chem.rev.,2018,118: 1917-; the sample was then analyzed for exosome particle size distribution and concentration using NanoSight in this study, as shown in figure 2(b), with exosomes having an average particle size of 106.7 ± 2.1nm, consistent with TEM results, at a concentration of 2.22 × 10⁹±2.39×10⁸Per mL; the expression conditions of the marker protein CD63 and the detection protein PSMA with prostate cancer diagnostic value in an exosome sample are detected by using Western blot as shown in a figure 2(c), the extracted LNCaP cell protein is selected as a positive control, the sample loading amount of each sample well is 20 mu g based on a BCA protein quantitative method, the exosomes from the LNCaP prostate cancer cell line can be seen to express the two proteins, and an experimental basis is provided for the next research.

2) Super-resolution microscope imaging verification of expression of CD63 and PSMA on exosome membranes

According to the report of the literature, the marker protein CD63 and the detection protein PSMA with prostate cancer diagnosis value in the exosome sample are membrane proteins, and to further confirm the conclusion, the topic uses the super-resolution imaging technology to characterize the co-localization of CD63 and PSMA and exosome membrane, so as to verify the expression of CD63 and PSMA on the exosome membrane derived from LNCaP prostate cancer cell line. As shown in fig. 3(a), the green fluorophore is CD36-FITC treated exosome, CD63 can bind to exosome membrane protein CD63, the diameter of exosome (green fluorophore) is shown to be around 100nm, and the diameter size of exosome according to NTA characterization is shown in the figure, fig. 3(b) is used for treating exosome membrane protein PSMA with red fluorescent dye Alexa Fluor 647, fig. 3c is an effect diagram after exosome membrane channel and exosome membrane protein channel image fusion, and the co-localization of exosome membrane and exosome membrane proteins (green fluorescence and red fluorescence) confirms the expression of CD63 and PSMA on exosome membranes from LNCaP prostate cancer cell lines.

3) Feasibility verification of nucleic acid aptamer-based 'Turn-on' type exosome nano fluorescence sensing technology

In order to verify the feasibility of the nano fluorescence biosensor technology, 1 mu M of PSMA aptamer and prostate cancer LNCaP cell-derived exosome are incubated for 15min at 37 ℃,3.2nM TPE-TA molecules are added to react for 10min in a dark mode, 32 mu g/mL GO is added to adsorb free aptamer and TPE-TA molecules, fluorescence signal detection is carried out on a fluorescence spectrophotometer, and meanwhile, 3.2nM TPE-TA pure solution, 3.2nM TPE-TA solution added with 1 mu M of PSMA aptamer and 32 mu g/GO are respectively subjected to fluorescence detection at a wavelength of 480nM by using the fluorescence spectrophotometer. As shown in FIG. 4, the fluorescence response of TPE-TA pure solution (curve d in FIG. 4) is the lowest; in the graph after the PSMA aptamer is added into the TPE-TA pure solution (curve a in FIG. 4), the fluorescence response value is increased sharply; after addition of the fluorescence quenching group GO (curve c of fig. 4), the fluorescence response dropped to near baseline levels; when PSMA (+) exosomes were present in solution (curve b of FIG. 4), the aggregated TPE-TA complexes could not be quenched by GO due to efficient binding of the aptamer to exosomes, and the fluorescence response value was significantly increased, with a signal-to-noise ratio (S/N) of about 6 times as compared to the control (curve c of FIG. 4).

4) Screening and verification of PSMA (+) exosome subgroup specific PSMA aptamer

Comparing the fluorescence signal to noise ratio (S/N) data of four PSMA aptamers in a "Turn-on" type fluorescence reporter system based on TPE-TA and GO after reaction with prostate cancer LNCaP cell-derived exosomes, as shown in fig. 5(a) - (d), it was found that PSMA aptamer P1 identified the highest fluorescence signal to noise ratio (S/N) of LNCaP exosomes under the same conditions (fig. 6 (a)). The secondary structure and melting temperature of four PSMA aptamers (P1, P2, P3, P4) were predicted using the online software NUPACK. As shown by analyzing the DNA sequence and the structure of four PSMA aptamers, the number of DNA bases of PSMA aptamer P1 is the largest, the secondary structure is the most complex, and therefore, more TPE-TA molecules can be accommodated to be gathered on the aptamer sequence, and the intensity of the emitted fluorescence signal is higher. Thus, this experiment was intended to select P1 as the optimal PSMA aptamer.

Using 1. mu.M of optimal PSMA aptamer P1 and varying concentrations (1X 10)⁷,1×10⁸,1×10⁹particles/. mu.L) reaction products of LNCaP exosomes from prostate cancer were subjected to PAGE electrophoresis, as shown in FIGS. 6(a) and 6(b), and the results showed P1 at different concentrations (1X 10)⁷,1×10⁸,1×10⁹particles/. mu.L) prostate cancer LNCaP exosomes the amount of free P1 remaining after reaction varied, and as the number of LNCaP exosomes increased, the amount of free P1 decreased, thus indirectly demonstrating the ability of PSMA aptamer P1 to recognize prostate cancer LNCaP cell-derived exosomes.

5) Study of reaction conditions

The important reaction conditions involved in the experiment mainly include TPE-TA concentration, GO concentration, reaction temperature and reaction time. In this regard, we have designed a series of experiments to observe the detection effect of the method under different reaction conditions to find the optimal reaction conditions for optimal detection performance.

The AIE nano molecule TPE-TA is used as a fluorescence signal group, and the reaction concentration of the AIE nano molecule TPE-TA directly influences the signal to noise ratio of the fluorescence response value of the nano fluorescence biosensor. To investigate the optimum reaction concentration of TPE-TA, a blank control group (no exosome added) and an experimental group (1X 10) were set up for the experiment⁸particles/. mu.L exosomes) under other reaction conditionsThe effect of a series of different TPE-TA response concentrations (1.6nM,3.2nM,4.8nM,6.4nM) on the fluorescence response and signal-to-noise ratio of the two groups was observed as a function. As shown in fig. 7(a), as the concentration of TPE-TA increases, the fluorescence response values of the control group and the experimental group both increase, because TPE-TA molecules have multiple positive charges and can emit high-intensity fluorescence after being combined with free PSMA aptamers in solution by electrostatic attraction, and under the condition that other conditions are not changed, the fluorescence response values are in direct proportion to the number of TPE-TA molecules combined on free PSMA away from the surface of GO in solution, so the fluorescence response values of the control group and the experimental group increase with the increase of the concentration of TPE-TA; the fluorescence response value signal to noise ratio is the ratio of the fluorescence response values of the experimental group and the control group, and reflects the capability of the method for distinguishing the existence of the target exosome, when the concentration of TPE-TA is 3.2nM, the fluorescence response value signal to noise ratio reaches the maximum value, which shows that when the concentration of TPE-TA is more than 3.2nM, although the fluorescence response value of the experimental group still increases along with the increase of the concentration of TPE-TA, the increase of background signals is more obvious because the TPE-TA on the free PSMA aptamer in the control group solution aggregates more, and the high concentration of TPE-TA can cause the excessive TPE-TA to agglomerate in the solution to emit light, and the fluorescence response value signal to noise ratio is reduced. Therefore, 3.2nM was chosen as the optimum response concentration for TPE-TA.

GO is used as a fluorescence quenching group, and the reaction concentration in the experiment also directly influences the signal-to-noise ratio of the fluorescence response value of the nano fluorescence biosensor. To investigate the optimal GO concentration for the reaction, a blank (no exosomes added) and experimental groups (10 additions) were set up⁸Individual prostate cancer exosomes), the effect of a series of different GO reaction concentrations (16 μ g/mL,24 μ g/mL,32 μ g/mL,40 μ g/mL) on fluorescence response values and signal-to-noise ratios was observed with other reaction conditions unchanged. As shown in fig. 7(b), as GO concentration increases, the fluorescence response values of the control group and the experimental group both decrease, because GO has very high quenching ability for various fluorophores, and under the condition of no change of other conditions, the fluorescence response values are inversely proportional to GO concentration in the solution, so that the fluorescence response values of the control group and the experimental group decrease as GO concentration increases; however, when the GO concentration is 32 mug/mL, the signal-to-noise ratio of the fluorescence response value reaches the maximum value, which shows that when the GO concentration is more than 32 mug/mL, although the GO concentration is higher than 32 mug/mLHowever, the fluorescence response value of the experimental group still decreases with the increase of the concentration of GO, but the quenching capacity of the GO of the control group cannot increase with the concentration, so that the background signal cannot continue to decrease, and the signal-to-noise ratio of the fluorescence response value is reduced. Therefore, choosing 32 μ g/mL for the optimal reaction concentration of GO can further reduce background signal and increase the sensitivity of the assay.

The structure, the recognition capability and the reaction rate of the aptamer can be changed by temperature, so that the sensitivity and the specificity of the reaction are influenced. To investigate the effect of different reaction temperatures on the reaction results, blank control (no target exosome added) and experimental groups (10 added) were set up⁸Individual target exosomes) under otherwise identical reaction conditions, the effect of a series of different temperatures (25 ℃,37 ℃,42 ℃) on the fluorescence response value and signal-to-noise ratio was observed. As shown in FIG. 7(c), the signal-to-noise ratio of the fluorescence response value reached a maximum value at a reaction time of 37 ℃, and this result indicates that: under the environment with too low temperature, the identification capability and the reflection efficiency of the aptamer are influenced due to insufficient energy; when the ambient temperature is too high, the aptamer participating in the reaction may form an unstable secondary structure, thereby affecting the fluorescence response value and stability. Therefore, the optimal reaction temperature for the cancer exosome detection method was determined to be 37 ℃.

The optimal reaction time not only can enable the detection method to make effective response to the target, improve the sensitivity of the method to the maximum extent, but also can shorten the detection time to the maximum extent. To examine the effect of different reaction times on the reaction results, blank control (no target exosome added) and experimental group (10 added) were set up⁸Individual target exosomes) under the condition that other reaction conditions are not changed, observing the influence of a series of different reaction times (10min,15min,20min,30min,45min,60min and 75min) on the fluorescence response value and the signal-to-noise ratio. As shown in FIG. 7(d), the fluorescence response value reached a maximum value when the reaction time was 15 min. Then, as the reaction time increases, the fluorescence response value does not increase or decrease inversely, which may be related to the fluorescence quenching of TPE-TA under the irradiation of exciting light in the solution for a long time. Therefore, the optimal reaction time of selecting 15min as the method can ensure the maximization of the fluorescence response value and improve the detection efficiencyRapid and sensitive detection of the PSMA (+) exosome subpopulation is now possible.

6) Specificity test

The diversity of the protein biomarkers of exosome subpopulations makes the enumeration of exosomes of a single subpopulation more complex. In the present study, the analysis and detection of the exosome subpopulation was based on the specific recognition of PSMA (+) exosomes by PSMA aptamers, and therefore, the method has excellent specificity. In order to evaluate the specificity of the nano fluorescence sensing technology for detecting the PSMA (+) exosome subgroup of the prostate cancer cells, under the optimal reaction condition, the detection method is used for carrying out fluorescence detection on LNCaP exo, and simultaneously recording the fluorescence response signal-to-noise ratios of four control groups of breast cancer cell exosome (MDA-MB-231exo), normal hepatocyte exosome (HL-7702exo), mouse normal macrophage exosome (RAW264.7exo) and normal human Plasma exosome (Plasma exo), and the experiment is repeated three times to evaluate the specificity of the detection method for detecting the PSMA (+) exosome (the experimental result is shown in figure 8). Before the experiment, the concentrations of LNCaP exo, MDA-MB-231exo, HL-7702exo, RAW264.7exo and Plasma exo were measured to be 3.66X 10, respectively, using NTA⁸、2.22×10⁸、4.22×10⁸、2.86×10⁸、1.08×10⁸particles/. mu.L. After appropriate dilution, the exosome concentration in each sample was made to be 1 × 10⁸particles/. mu.L. The research result shows that the signal-to-noise ratios of the fluorescence detection response values of the four groups of control groups are less than 1, while the signal-to-noise ratios of the fluorescence detection response values of the LNCaP exosomes are greater than 5 and are obviously higher than those of the other four groups of fluorescence detection signals. It is worth mentioning that the signal-to-noise ratio of the fluorescence detection response value of the exosome derived from the normal human plasma is only one tenth of the signal-to-noise ratio of the fluorescence response value of the exosome derived from the prostate cancer cell. The result shows that the exosome subgroup detection method established based on the nano fluorescence sensing technology can effectively distinguish the prostate cancer LNCaP cell source PSMA (+) exosome subgroup from other tumor cells or normal cell source exosomes, can effectively avoid the interference of the normal cell source exosomes in plasma on the detection result, and has good specificity.

7) Sensitivity test

To evaluate the nano-fluorescence basedSensitivity of a detection method established by a sensing technology on the detection of the PSMA (+) exosome subpopulation derived from the prostate cancer cells, and the method is used for detecting a series of PSMA (+) exosome subpopulations derived from the prostate cancer LNCaP cells with different concentrations. We performed gradient dilution (1.83X 10) of prostate cancer cell-derived exosomes⁷,1.53×10⁷,1.22×10⁷,6.10×10⁶,3.66×10⁶,1.22×10⁶，6.10×10⁵,4.07×10⁵particles/. mu.L) as target substance for the system, these different concentrations of exosomes (group 3 parallel assays) were analyzed with this detection method under optimal reaction conditions (37 ℃,15 min). From the collected fluorescence spectrogram, the fluorescence intensity collected at the position with the strongest emission light is correspondingly enhanced along with the increase of the exosome concentration, namely, the fluorescence intensity collected in the range is in positive correlation with the exosome concentration. According to the fluorescence spectrum chart 9(a), the fluorescence signals collected at 480nm of each experimental group are subjected to statistical analysis and linear fitting to obtain a chart 9 (b): at an exosome concentration of 4.07X 10⁵-1.83×10⁷Fluorescence intensity is proportional to the number of tumor cells in the region of particles/. mu.L; the mathematical model for detecting the PSMA (+) exosome subgroup from the prostate cancer cells by the method can be obtained by linear fitting, the linear equation is that y is 1.139x +51(y is fluorescence intensity, and x is concentration of the PSMA (+) exosomes), and the correlation coefficient is 0.994; estimating detection limit according to the blank signal plus 3 times of standard deviation, and calculating to obtain the minimum detection limit of 3.43 × 10⁵particles/μL。

2.2.12 evaluation of clinical test Performance

In order to further verify whether the constructed method has analytical feasibility and clinical application potential, 20 prostate cancer patients and 7 healthy human plasma samples were collected clinically, examined by the southern medical university hospital ethics committee, and informed consent was obtained from all subjects and families. The diagnosis of prostate cancer patients meets the criteria of Chinese guidelines for urological disease diagnosis and treatment (2014 edition).

500 μ L of plasma was pre-treated by centrifugation at 10,000g for 30min, centrifuged at 54,000rpm for 2h in an ultracentrifuge, collected and then resuspended in PBS, centrifuged under the same conditions, and plasma-derived exosomes were collected. NTA is adopted to measure the concentration of plasma exosome samples, and after dilution, the same exosome concentration is taken for subsequent experiments. Under the optimal reaction condition, the detection method is used for carrying out fluorescence detection on the plasma source exosomes of the prostate cancer patient group and the healthy human group, and the experiment is repeated three times to evaluate the clinical detection performance of the detection method. The results are shown in fig. 10, and the comparison result of the fluorescence intensity of the detection of the plasma-derived exosomes of the prostate cancer patient group and the healthy human group according to the detection method shows that the concentration of the PSMA (+) exosomes of the prostate cancer patient is significantly higher than that of the healthy human.

In conclusion, the invention not only proves that PSMA on the exosomes derived from human plasma can be used as a biomarker for identifying healthy subjects and prostate cancer patients, but also further proves that the method can effectively avoid the influence of exosomes derived from normal cells in a clinical plasma sample on the detection result, has good anti-interference capability and has great clinical application potential in the aspect of prostate cancer PSMA (+) exosome subgroup analysis.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

SEQUENCE LISTING

<110> southern hospital of southern medical university

<120> tumor exosome nano fluorescence detection kit and application thereof

<130> PCQNF193198

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Claims (7)

1. A kit for tumor exosome nano fluorescence detection is characterized in that: the fluorescence detection kit comprises an aptamer, a fluorescence reporter group and a fluorescence quenching group, wherein the nucleotide sequence of the aptamer contains any one of sequences shown as SEQ ID NO.1-SEQ ID NO.3, the fluorescence reporter group is selected from aggregation-induced emission molecules, the aggregation-induced emission molecules are selected from tetraphenylethylene derivatives, and the fluorescence quenching group is selected from graphene oxide.

2. The kit of claim 1, wherein: the tetraphenylethylene derivative is at least one selected from TPE-TA, [12] aneN 3 modified tetraphenylethylene compound, TPE-Py and TPE-Z.

3. The kit of claim 1, wherein: the graphene oxide is selected from single-layer graphene oxide.

4. The kit of claim 3, wherein: the thickness of the single-layer graphene oxide is 0.8-1.2 nm.

5. The kit of claim 1, wherein: also includes a buffer solution.

6. The kit of claim 5, wherein: the buffer solution is PBS buffer solution.

7. Use of a kit according to any one of claims 1 to 6 for the preparation or screening of a medicament for the diagnosis and/or prevention and/or treatment of prostate cancer, for the in vitro diagnosis and/or risk stratification of prostate cancer.

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