阅读说明：本技术 一种基于单颗粒成像的外泌体亚型分析方法 (Exosome subtype analysis method based on single particle imaging ) 是由 余辉 翟春荟 杨玉婷 于 2021-06-08 设计创作，主要内容包括：本发明涉及一种基于单颗粒成像的外泌体亚型分析方法,该方法通过对外泌体单颗粒的动态成像和数字化分析,同时获取粒径及表面分子标志物信息,通过数据分析算法进行亚型的分类。首先,在金属芯片表面修饰识别分子,通过流体装置将该待测外泌体样品加入金属芯片表面的反应腔,通过高灵敏的显微成像系统及数据分析软件实现外泌体与分子相互作用过程的动态检测,构建外泌体粒径与分子标志物间的定量模型,实现亚型的聚类分析,解决了外泌体亚型分析中亚型样品分离困难的关键问题。(The invention relates to a single-particle imaging-based exosome subtype analysis method, which is used for dynamically imaging and digitally analyzing exosome single particles, simultaneously acquiring particle size and surface molecular marker information and classifying subtypes through a data analysis algorithm. Firstly, modifying recognition molecules on the surface of a metal chip, adding the exosome sample to be detected into a reaction cavity on the surface of the metal chip through a fluid device, realizing dynamic detection of an interaction process of exosomes and molecules through a high-sensitivity microscopic imaging system and data analysis software, constructing a quantitative model between an exosome particle size and a molecular marker, realizing subtype cluster analysis, and solving the key problem of difficult subtype sample separation in exosome subtype analysis.)

1. A method for exosome subtype analysis based on single particle imaging, comprising the steps of:

1) bonding the PDMS channel with the gold sheet to obtain a chip, and forming a stable sample flow cell;

2) bonding a plurality of aptamers to the surface of the chip obtained in the step 1) by an Au-S bond self-assembly method, and blocking the remaining active sites on the surface of the chip by using MCH and BSA to obtain an exosome sensor chip based on aptamer capture to form a microfluidic sample injection system;

3) adding an exosome sample to be detected into the microfluidic sample injection system obtained in the step 2), and monitoring and recording the specific interaction of the exosome and the aptamer in real time by adopting a surface plasma resonance imaging platform without a label;

4) analysis was based on single particle counts and particle size statistics of image analysis.

2. The method for analyzing exosome subtype based on single-particle imaging according to claim 1, characterized in that in step 1), the PDMS channel is obtained by the following method: drawing a channel pattern by adopting CAD, printing the pattern on a film plate mould, forming a graphical microfluidic channel on the surface of a silicon wafer of the film plate mould by utilizing a photoetching technology, cleaning, and sequentially performing high-temperature treatment and silanization treatment to make the surface of the silicon wafer hydrophilic; and pouring the PDMS liquid into the mold, and curing at high temperature to obtain the PDMS channel.

3. The method according to claim 2, wherein the PDMS channel size: the length multiplied by the width multiplied by the height is 10mm multiplied by 1mm multiplied by 30 mu m, and each PDMS chip contains three channels;

the high-temperature treatment temperature is 120-150 ℃, and the time is 1-3 hours;

the curing temperature of the high-temperature curing is 85 ℃, and the curing time is 2-3 hours.

4. The method for analyzing exosome subtype based on single-particle imaging according to claim 1, characterized in that, in the step 1), the method for bonding PDMS channels and gold sheets comprises the following steps:

1-1) fully cleaning the Au chip, drying the Au chip by using nitrogen, and placing the Au chip in a clean culture dish for later use;

1-2) irradiating oxygen Plasma on the PDMS channel and the Au chip simultaneously, combining the PDMS channel and the Au chip, and curing at high temperature to obtain the sample flow cell containing the chip.

5. An exosome subtype analyzing method based on single particle imaging according to claim 4 characterized in that the time of irradiating oxygen Plasma in step 1-2) is 50 seconds; the bonding temperature of PDMS and Au chip is 120 ℃.

6. The exosome subtype analyzing method based on single particle imaging according to claim 1, characterized in that step 2) is specifically:

2-1) mixing the aptamer, the MCH and the TCEP to prepare a modification solution, adding the modification solution into the sample flow cell, and carrying out light-shielding overnight modification;

2-2) replacing the Aptamer modification solution with a BSA solution, standing to enable BSA to occupy unmodified binding sites on the surface of the Au chip, and then washing with PBS to obtain the Aptamer/MCH/BSA modified exosome capture sensing chip.

7. The method for analyzing exosome subtype based on single particle imaging according to claim 6, characterized in that in step 2-1), the aptamer is selected according to the type of the detected disease, wherein one end is sulfhydryl; aptamer, MCH to TCEP in a molar ratio of 1: 1: 5; the temperature for overnight modification in dark is 20 ℃;

in the step 2-2), the BSA solution has a mass volume concentration of 1% w/v, and after modification, the modified BSA solution is washed three times with PBS.

8. The exosome subtype analyzing method based on single-particle imaging according to claim 1, characterized in that the surface plasmon resonance imaging platform in step 3) adopts an objective lens which is an oil lens and has a numerical aperture range of 1.39-1.79;

the adopted laser light source is p-polarized light, and the wavelength range of the light source is 550-780 nm;

the pixel size range of the CMOS camera is 30-200 nm.

9. The exosome subtype analyzing method based on single particle imaging according to claim 1, characterized in that the specific analyzing method in step 4) is as follows:

4-1) identifying an exosome image on a single-frame picture by using an image difference algorithm based on a Python platform;

4-2) tracking the attachment dissociation process of a single exosome by using a tracking algorithm according to a time sequence, and recording the maximum brightness of the exosome in the attachment process as a characteristic numerical value of the particle size;

4-3) dividing the exosome population into three subgroups according to the particle size, and respectively counting the number ratio in each subgroup.

10. The exosome subtype analyzing method based on single particle imaging according to claim 9, characterized in that in step 4-3), silica nanoparticles of 30, 50, 100 and 160nm are used as standard substances, a standard curve is drawn, and the particle size of exosome is measured.

Technical Field

The invention belongs to the technical field of in-vitro diagnosis, and relates to a separation-free exosome subtype analysis method based on surface plasmon resonance imaging.

Background

Liquid biopsy (Liquid biopsy) technology is used for early screening, guiding treatment schemes, monitoring treatment and monitoring recurrence of cancer patients by detecting circulating biomarkers and the like in blood, and is one of the most promising cancer diagnosis and treatment tools at present. Exosomes are micro-membrane vesicles secreted by cells and having diameters of 30-150 nanometers, and are widely present in most human body fluids, such as urine, blood, milk, saliva and the like. Exosomes contain specific proteins, lipids and nucleic acids in their host cells, can be transmitted to other cells as signal molecules to alter the functions of other cells, and play important roles in a variety of physiopathological processes, such as antigen presentation in immunity, tumor growth and migration, repair of tissue damage, and the like. Exosomes secreted by different cells have different compositions and functions and therefore can serve as biomarkers for fluid biopsies. Compared with other circulating biomarkers, the exosome has the characteristics of easy enrichment, strong stability and difficult degradation, and is paid more and more attention.

Due to the differences in tumor types, tumor mutations, cell sources, and extracellular environment, exosomes in blood have severe individual differences in size distribution, number, and composition of contents. By using an asymmetric fluid technology, the exosome population of 30-150nm can be further divided into three subgroups of less than 50nm, 60-80nm and 90-120nm according to particle size. Mass spectrometry analysis revealed that each subpopulation had its own signature markers. This indicates that there is a specific relationship between the size of the exosomes and the content. Depending on the type of cancer, the exosomes secreted by cancer cells carry cancer-specific markers and are also associated with particle size. The current liquid biopsy technology based on exosome population analysis, such as thermophoresis enrichment technology combined with appropriate ligand fluorescence multiparameter detection, has low accuracy for diagnosis of 6 common cancers. Therefore, the development of exosome subtype analysis methods related to particle size and surface markers is of great importance for the accurate diagnosis of cancer.

Current technologies that enable exosome analysis include: the size and the form of the exosome are analyzed by an electron microscope, the average particle size is estimated by Dynamic Light Scattering (DLS), and the protein molecular marker is detected by methods such as Westernblot and ELISA. In the whole analysis process, not only is sample processing complex and time consuming, but also the sample consumption is large, and meanwhile, difference information among exosome individuals is lacked, and the particle size and molecular marker information cannot be obtained simultaneously. The Yangxi Meiji professor topic group at Xiamen university makes outstanding achievements in the aspect of NanoFCM technology, realizes high-sensitivity measurement on 40nm exosomes, but lacks enough molecular information, and needs to realize specific detection by carrying out fluorescence immunolabeling on an exosome sample, so the Yangxi Meiji professor is limited by the number of fluorescence channels, fluorescence analysis luminous efficiency, sample pretreatment, semi-quantitative detection and the like, and has great limitation in the aspect of exosome subtype analysis. However, the traditional SPR technology cannot simultaneously obtain the particle size and molecular marker information of exosome and even analyze the subtype of exosome due to lack of single particle imaging analysis capability. The way in which exosome populations are grouped by particle size by asymmetric fluidic techniques or size exclusion chromatography techniques is limited by rigorous technical means and is extremely impractical in the clinic. Therefore, there is an urgent need to develop a separation-free exosome subtype analysis technique in relation to size and surface markers.

Disclosure of Invention

The invention aims to overcome the difficulty that exosomes need to be further separated according to the particle size in the prior art, and provides a separation-free exosome subtype analysis method on a single exosome scale, which has the characteristics of high sensitivity, high specificity and simplicity in operation.

The purpose of the invention can be realized by the following technical scheme: a method for exosome subtype analysis based on single particle imaging, the method comprising the steps of:

1) bonding the PDMS channel with the gold sheet to obtain a chip, and forming a stable sample flow cell;

2) bonding a plurality of aptamers to the surface of the chip obtained in the step 1) by an Au-S bond self-assembly method, and blocking the remaining active sites on the surface of the chip by using MCH and BSA to obtain an exosome sensor chip based on aptamer capture to form a microfluidic sample injection system;

3) adding an exosome sample to be detected into the microfluidic sample injection system obtained in the step 2), and monitoring and recording the specific interaction of the exosome and the aptamer in real time by adopting a surface plasma resonance imaging platform without a label;

4) analysis was based on single particle counts and particle size statistics of image analysis.

In step 1), the PDMS channel is obtained by the following method: drawing a channel pattern by adopting CAD, printing the pattern on a film plate mould, forming a graphical microfluidic channel on the surface of a silicon wafer of the film plate mould by utilizing a photoetching technology, cleaning, and sequentially performing high-temperature treatment and silanization treatment to make the surface of the silicon wafer hydrophilic; and pouring the PDMS liquid into the mold, and curing at high temperature to obtain the PDMS channel.

Further, the PDMS channel size: the length multiplied by the width multiplied by the height is 10mm multiplied by 1mm multiplied by 30 mu m, and each PDMS chip contains three channels;

the temperature of the high-temperature treatment is 120-150 ℃, and the time is 1-3 hours;

the silanization treatment comprises the following steps: placing the silicon wafer on a piece of filter paper, taking 5 microliters of silanization reagent (3-Mercaptopropyltrimethoxysilane (MPTS)), dripping on the filter paper in a ventilation kitchen, buckling the filter paper and the silicon wafer by using a culture dish to volatilize the silanization reagent, and finishing the silanization process after 5-10 min.

The PDMS liquid is a commercial product, for example, Dow Corning 184 polydimethylsiloxane PDMS can be selected, and the PDMS liquid is prepared by mixing Part A and Part B according to a mass ratio of 10: 1, wherein Part A is a polydimethylsiloxane prepolymer, and Part B is a prepolymer with a vinyl side chain and a crosslinking agent.

The curing temperature of the high-temperature curing is 85 ℃, and the curing time is 2-3 hours.

Further, in step 1), the method for bonding the PDMS channel and the gold plate includes the following steps:

1-1) fully cleaning the Au chip, drying the Au chip by using nitrogen, and placing the Au chip in a clean culture dish for later use;

1-2) irradiating oxygen Plasma on the PDMS channel and the Au chip simultaneously, combining the PDMS channel and the Au chip, and curing at high temperature to obtain the sample flow cell containing the chip.

Further, the time for irradiating oxygen Plasma in the step 1-2) was 50 seconds; the bonding temperature of PDMS and Au chip is 120 ℃.

Further, the step 2) is specifically as follows:

2-1) mixing the aptamer, the MCH and the TCEP to prepare a modification solution, adding the modification solution into the sample flow cell, and carrying out light-shielding overnight modification;

2-2) replacing the Aptamer modification solution with a BSA solution, standing to enable BSA to occupy unmodified binding sites on the surface of the Au chip, and then washing with PBS to obtain the Aptamer/MCH/BSA modified exosome capture sensing chip.

Further, in step 2-1), the aptamer is selected according to the type of the disease to be detected, wherein one end is a thiol group; aptamer, MCH to TCEP in a molar ratio of 1: 1: 5; the temperature for overnight modification in dark is 20 ℃; the microfluidic sample injection system comprises a micro-injection pump and a microfluidic pipeline, wherein the microfluidic pipeline comprises a PDMS channel, the PDMS channel is bonded with a gold sheet, and a characteristic aptamer is modified on the gold sheet.

In the step 2-2), the BSA solution has a mass volume concentration of 1% w/v, and after modification, the modified BSA solution is washed three times with PBS.

Further, the surface plasma resonance imaging platform in the step 3) adopts an oil lens as an objective lens, and the numerical aperture range of the objective lens is 1.39-1.79;

the adopted laser light source is p-polarized light, and the wavelength range of the light source is 550-780 nm;

the pixel size range of the CMOS camera is 30-200 nm.

Further, the specific analysis method in the step 4) comprises the following steps:

4-1) identifying an exosome image on a single-frame picture by using an image difference algorithm based on a Python platform;

4-2) tracking the attachment dissociation process of a single exosome by using a tracking algorithm according to a time sequence, and recording the maximum brightness of the exosome in the attachment process as a characteristic numerical value of the particle size;

4-3) dividing the exosome population into three subgroups according to the particle size, and respectively counting the number ratio in each subgroup.

Further, in the step 4-3), silica nanoparticles of 30, 50, 100 and 160nm are used as a standard substance, a standard curve is drawn, and the particle size of exosome is measured.

Compared with the prior art, the invention has the following advantages:

(1) the invention can realize the full-particle-size analysis of the exosome with the particle size of 30-150nm by utilizing the iPM platform, monitors the specific interaction of the exosome and the surface aptamer in real time, and has the detection sensitivity of 5 multiplied by 10⁷Individual exosomes/mL, close to NTA capacity.

(2) The invention utilizes the aptamer as a recognition element, is directly combined to the surface of the Au chip by an Au-S bond self-assembly membrane method, has the advantages of simple steps, easy realization, no need of carrying out other complex modification treatment on the substrate and the like, can realize the specific combination of the target exosome in the detection process, and effectively improves the specificity of the sensor.

(3) The single particle counting and particle size statistical software system based on image analysis can simply and quickly process data, simultaneously obtains the particle size and surface marker information, avoids the technical difficulty of separation in exosome subtype analysis, and is expected to be used for exosome subtype analysis in blood liquid biopsy technology and accurately judging the type of cancer.

Drawings

FIG. 1 is a schematic diagram of a system for analyzing exosome subtypes based on surface plasmon resonance;

FIG. 2 is a particle size and brightness standard working curve drawn by iPM platform detection of 30-150nm silica nanoparticles;

FIG. 3 is an exosome concentration test of the iPM platform against a breast cancer cell line MCF-7 source;

figure 4a is a subtype analysis of particle size and surface markers in tumor-derived cell lines using the iPM platform: MCF-7 cell line particle size distribution;

figure 4b is a subtype analysis of particle size and surface markers in tumor-derived cell lines using the iPM platform: analyzing the proportion of subgroups of exosomes derived from four tumors;

FIG. 5 is a graph comparing the amount of exosome-captured by the aptamer sensor of the present invention with that of BSA modified chip.

Detailed Description

The present invention will be described in detail with reference to specific examples.

The invention discloses a method for detecting exosome groups extracted from cancer cell line culture solution by adopting an SPR chip modified by a nucleic acid aptamer, which comprises the following steps: injecting 10uL of exosome sample to be detected into a PDMS channel modified with a characteristic nucleic acid aptamer by adopting a microfluidic sample injection system, selecting a proper observation area with the flow rate of 2uL/min, recording an interaction image of the exosome and the aptamer in real time by utilizing an SPRM platform, dividing an exosome group into three subgroups according to standard particle size curves drawn by silica with the particle sizes of 30nm, 50nm, 70nm, 100nm and 160nm after about 2-5min, and realizing the cluster analysis of the subgroups. As shown in fig. 1.

And subsequently, analyzing the image by using an automatic software analysis system (namely a Python platform which is a common system in the field), removing background noise by using an image difference method to obtain a single-frame exosome image signal, analyzing the specific binding effect of the exosome and the surface-modified aptamer by using a time sequence, and extracting the brightness and quantity information of the exosome.

The specificity of the aptamer SPR sensor is measured by the following method: and respectively introducing the exosome sample solution from the cancer cell culture solution into the aptamer channel and the channel modified only by BSA, carrying out image acquisition and data processing under the same conditions by adopting the same method, analyzing the number of exosomes specifically adsorbed on the chip in the sample under the two modification conditions, and inspecting the specific performance of the aptamer SPR sensor.

An interference plasma Imaging (iPM) technology realizes dynamic label-free imaging of exosome single particles, the detection limit reaches 30 nanometers, and the particle size range of exosomes is completely covered; by carrying out molecular modification on the surface of the iPM chip, the quantitative detection of the molecular marker on the surface of the exosome can be simultaneously realized. Different from the traditional prism-based SPR/SPRi technology, the iPM technology adopts an objective lens with high magnification and large numerical aperture as a light path coupling element, and is matched with a high-performance camera to collect high-resolution exosome images, acquire image characteristics in a quantification mode, and establish quantitative relations between the image characteristics and information such as exosome particle size, exosome concentration and the like. The key technical principle is that an interference imaging theoretical model based on iPM is used for optimizing experimental conditions, so that the signal-to-noise ratio reaches the shot noise limit, and the imaging sensitivity is improved to a single-particle detection level of 30nm through numerical reconstruction and a time-space domain filtering algorithm; meanwhile, iPM combines the advantages of the traditional SPR biosensing technology, namely, high-throughput analysis of molecular markers can be realized through surface modification, and multi-parameter detection of physical and molecular information is realized on the platform.

Aptamer (aptamer), also known as an "artificial antibody", is a single-stranded DNA or RNA fragment obtained by in vitro screening by the exponential enrichment ligand phylogenetic evolution (SELEX) technique, generally 20-100 nucleotides in length, and can be specifically and tightly bound with a corresponding ligand, thereby realizing the specific detection of a target molecule. The aptamer is used as an identification unit, and has the advantages of small size, good affinity, easy artificial synthesis and modification, strong stability, simple fixation operation on a substrate, low detection limit when being used for detecting small molecules and the like. Therefore, the invention combines the interference plasma imaging technology with the nucleic acid aptamer with the specific recognition function to construct the sensor chip for capturing the tumor-derived exosome specifically, so as to obtain good detection sensitivity and specificity.

Specifically, the tumor molecule specific aptamer is self-assembled and modified on the surface of the iPM chip by utilizing an Au-S bond, and the remaining binding sites are blocked by utilizing MCH and BSA, so that the final sensor chip is obtained. The check aptamer is used as a capture element, the selectivity and specificity of the sensor are improved, the iPM platform is used for collecting data, the particle size information of a single exosome can be obtained, the ultrasensitive characteristic of the iPM is added, exosome particles with the particle size of 30nm can be observed, and the specific interaction information of the exosome membrane surface antigen and the nucleic acid aptamer can be observed by utilizing the characteristic of SPR. The detection method is rapid, simple and convenient, and can realize label-free single molecule detection of exosome groups.

The invention utilizes Python software to realize full-automatic analysis of data. By using the differential background removal method, the background noise can be effectively removed, so that the signal can be more effectively reflected. The exosome image is identified based on a Gan neural network, and the accuracy rate can reach more than 90%. The software system can identify standard silica particles with the particle size of 30nm, high-sensitivity particle identification is realized, and the data analysis efficiency is improved.

Aiming at the difficulty that the subpopulation needs to be separated in advance in the existing exosome analysis process, the invention combines an iPM detection platform with a nucleic acid aptamer with a specific recognition function to construct an SPR sensor for detecting a single exosome. Due to the surface near-field effect, the interference of a body solution in the detection process is effectively avoided, the sensitivity of detection is greatly improved by the iPM platform, and the whole particle size detection of the exosome of 30-150nm is realized; in addition, the platform has the performance of SPR, can realize the monitoring of the dynamic action of a single exosome and a specific aptamer, and realizes the specific detection. And the collected data can be rapidly analyzed by combining with efficient image processing software. The method has the advantages of simple chip manufacturing process, strong operability and good reproducibility, can simultaneously realize the detection of the particle size and the surface marker, and can analyze exosome subgroups to obtain a more accurate cancer identification method.

Example 1:

the preparation method of the aptamer iPM sensor for detecting the exosome comprises the following steps:

(1) and (5) processing the chip. Washing 2.0cm multiplied by 2.0cm BK-7 glass slide with deionized water, removing large particle dust on the surface, cleaning with absolute ethyl alcohol, washing off partial organic stains on the surface, repeating the steps for three times, drying the water stains and the ethyl alcohol solvent on the surface with nitrogen, and cleaning until no residual stains visible to naked eyes exist on the surface. And finally, burning the surface by using hydrogen flame to improve the flatness and further remove organic matter residues on the surface. A layer of metal chromium with the thickness of 3nm is plated on the surface of the magnetic control sputtering FHR instrument, so that the adhesive force between the metal chromium and a gold film is improved. And then plating a 47nm gold layer on the chromium surface, and finishing the primary processing of the chip. The chips were stored in clean storage boxes.

(2) And (4) chip pretreatment. And taking out a chip to be modified from the glass box, cleaning the surface by using deionized water and absolute ethyl alcohol in sequence, and repeating the steps for three times until the surface is free from macroscopic stains. And (3) drying the surface by using clean nitrogen, and then cleaning the surface back and forth by using hydrogen flame to further remove organic matters on the surface. Note that the flame does not make contact with the gold film for a long time, otherwise the chip is destroyed.

(3) And (5) constructing a micro-fluid sample injection system. The PDMS channel processing process is as follows: drawing a pattern of a channel by CAD, wherein the dimension is 10mm multiplied by 1mm multiplied by 30 mu m (length multiplied by width multiplied by height), each PDMS chip comprises three channels, manufacturing a film plate mould, utilizing the photoetching technology to pattern a template of a microfluidic channel on the surface of a silicon chip, and then cleaning, carrying out high-temperature treatment and silanization treatment to change the surface of the silicon chip into hydrophilic; will be as follows 10: 1(Part A: Part B), and curing at high temperature for 2-3 hours to obtain the PDMS channel. Irradiating the prepared PDMS channel and the Au chip with oxygen Plasma for 50 seconds simultaneously, combining the PDMS channel and the Au chip, and curing at high temperature to obtain a sample adding system;

(4) and (3) preparing the aptamer modification liquid. The selection of the aptamer corresponds to the molecular marker of the tumor to be detected. One end of the aptamer is provided with a sulfydryl and is used for self-assembling a monomolecular layer with the surface of the gold film. Taking 1 mu L of aptamer mother liquor (500 mu M), 1 mu L of MCH mother liquor (500 mu M) and 5 mu L of TCEP mother liquor (500 mu M), adding the mixture into 93 mu L of 1 XPBS solution, fully shaking and uniformly mixing, and standing for 30min at room temperature to obtain the chip surface modification solution.

(5) Construction of specific aptamer sensors. And (3) taking 20 mu L of the aptamer modification solution into a micro-syringe, slowly introducing the micro-syringe into a PDMS channel by using a micro-injection pump, and placing the micro-syringe in a room temperature and dark for overnight modification. And (5) incubating for 12h to prepare a specific aptamer sensing interface. After the modification, the modified solution was discharged, washed three times with PBS, and then the remaining binding sites were blocked with 20 μ L of 1% BSA solution to avoid nonspecific adsorption of exosomes on the gold membrane, and finally washed with 1 × PBS (pH 7.4) to prepare an aptamer sensor.

Example 2:

detection of silica standards was performed using the specific aptamer sensor prepared in example 1.

Use baseSPRM reconstructed in commercial Total internal reflection microscope (Olympus IX-81) as detection instrument, oil lens with magnification of 60 times and numerical aperture N.A ═ 1.49 was selected as light source, the light source was ultra-wideband light source SLED, the intensity of light source control current was 150mA, and the observation field was 512 × 512pixels (full field of view:51.2 × 51.2 μm)²). The imaging signal of SPRM is recorded by controlling a CCD camera (Photometrics) by a micro-manager. Cell lens software is used for controlling the incident angle of the light path.

Preparing silica nanoparticle standard substances with appropriate concentrations, wherein the particle diameters are respectively 30nm, 50nm, 70nm, 100nm and 160 nm. And (3) respectively introducing the standard substance into the microfluidic channels containing the aptamer sensors obtained in the example 1, and performing data acquisition at a sampling rate of 100FPS to obtain a particle size and brightness standard curve of the silicon dioxide standard substance for calibrating the particle size of the exosome sample. Due to the high-sensitivity detection characteristic of the iPM platform, a signal of 30nm silicon dioxide can be detected. As shown in FIG. 2, the detected characteristic brightness increases with increasing particle size, which is an exponential relationship.

Example 3:

detection of exosome samples was performed using the specific aptamer sensor prepared in example 1.

Exosome samples of full-size in cell culture broth were extracted by differential centrifugation, exosome pellets were dissolved in 50 μ L of 1 × PBS (pH 7.4) and stored in a refrigerator at 4 ℃ for later use. Before the sample is tested, NTA is used to measure the concentration, and the concentration of the sample is adjusted to 10⁹And (4) mixing the exosomes/mL by ultrasonic oscillation, and then using the exosomes/mL for detection. The microfluidic channel was filled with 1 × PBS (pH 7.4) solution, and the angle of the incident angle was adjusted to be slightly larger than the SPR resonance angle. The field of view was observed with left and right movements, and a clear surface field was sought for exosome determination.

The linear concentration range of the method was first tested. Respectively at a concentration of 10⁷，5×10⁷，10⁸，10⁹And (3) introducing each exosome/mL sample into the testing channel, counting the number of exosomes, and drawing a leading curve, as shown in figure 3.

Taking 10 mu L of the exosome sample to be detected, injecting the exosome sample into a PDMS pipeline by using a micro-injection pump, and passing through the area to be detected at the flow rate of 1-5 mu L/min according to the experimental condition. Meanwhile, sampling is carried out at a sampling rate of 100FPS, tumor specific aptamers such as CD63, HER2, EpCAM, PTK7, PSMA and PD-L1 are respectively modified on the surface of a breast cancer cell line MCF-7 as an example, and exosomes derived from the MCF-7 cell line are specifically detected. Recording the binding condition of the exosome on the surface of the specific aptamer, and acquiring the characteristic brightness and the specific binding condition of the single-particle exosome by using a software processing system. As shown in FIG. 4a, the particle size distribution of MCF-7 specific binding exosomes on various surfaces can be derived. As shown in FIG. 4b, subpopulation information of MCF-7 specific binding exosomes of different particle size ranges can be derived.

Example 4:

an exosome-sample-specific assay was performed using the aptamer sensor prepared in example 1:

in the process of chip modification, the specific aptamer is replaced by BSA, and chip modification is carried out to serve as a negative control. The sample preparation, data collection, and data analysis processes were performed using the same procedures as in example 3. The amount of binding on the BSA surface was compared to that on the specifically modified HER2 aptamer surface to demonstrate specific capture of exosomes by the specific aptamer chip. As shown in FIG. 5, it can be seen that the capture amount is higher in the exosome-sample-specific detection performed in the aptamer sensor of the present invention.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.