CN111999270A - An exosome detection method based on whispering gallery mode optical microcavity - Google Patents

Abstract

A method for detecting intracellular substances by detecting cell secretions based on a whispering gallery mode belongs to the cross field of laser technology and biomedicine. The method includes the following points: (1) the selection of the whispering gallery microcavity; (2) the selection of the surface treatment of the microcavity; (3) the identification and detection method of the microcavity and the exosomes secreted by the cells to be tested. The invention has high detection sensitivity and can realize high-efficiency identification and detection outside the cell. The whispering gallery mode makes the fluorescence resonate in the cavity to generate laser light, which realizes the function of prolonging the tracking detection time and realizing the tracking and identification function according to the enhanced detection signal.

Description

An exosome detection method based on whispering gallery mode optical microcavity

technical field

The invention is a microcavity of the whispering gallery mode to detect cell secretion exosomes. The technology mainly involves the principle of cell laser and the detection and identification method of exosomes and microcavities, belonging to laser technology and biomedical photonics. field.

Background technique

Whispering gallery mode optical microcavity (microsphere cavity, microring cavity, microdisk cavity, etc.) has excellent characteristics such as extremely small mode volume, ultra-high Q value, ultra-high energy density and extremely narrow linewidth, so it has become the most advanced optical microcavity. One of the optical devices with development potential. As a special optical resonator, when the laser is coupled into the microcavity, and under the conditions of total reflection and phase matching, the optical signal is continuously and totally reflected by the boundary in the microcavity, and the photons are localized for a long time. When the gain is greater than the loss under external excitation, a new waveband laser signal will be output.

Optical microcavities are widely used in biological sensing and detection due to their small size and sensitivity. The detection principle is that light is coupled into the microcavity to satisfy the resonance condition, and a total reflection occurs on the surface of the microcavity to form an evanescent wave of about 200 nm. The refractive index will also change. Different substances cause different changes in the refractive index. At this time, the existence of the object to be tested can be determined by the shift of the spectrum.

Exosomes are cell secretions, extracellular vesicles surrounded by lipid bilayers, 20-170 nm in size, and contain proteins and nucleic acids. In addition to self-carried marker proteins and tissue- or cell-type-specific proteins that reflect cell origin, it also contains various mRNAs, microRNAs, and siRNAs. They are secreted by all cells and widely present in body fluids, including blood, tears, saliva, urine, breast milk, cerebrospinal fluid, and abdominal fluid. Therefore, the detection and discovery of exosomes is helpful for the exploration and discovery of human life.

When the total reflection of the light beam on the inner wall of the microcavity travels around an optical path that is an integer multiple of the wavelength, a resonance phenomenon will occur, confining the resonant photons to the micrometer scale for a long time, which means that there are more substances and photons within the resonant mode range. interaction occurs. In 2016, Frank Vollmer's group successfully detected single atomic ions in aqueous solution by shifting the whispering gallery resonance frequency based on the microsphere cavity of the whispering gallery mode and the plasmon resonance coupling of gold nanorods.

The present invention is based on this background.

SUMMARY OF THE INVENTION

The invention is a method for cell exploration by detecting cell secretion exosomes based on the whispering gallery mode, which belongs to the cross field of laser technology and biomedicine. The method includes the following points: (1) the selection of the whispering gallery microcavity; (2) the surface modification selection of the microcavity; (3) the identification and detection method of the microcavity and the cell exosomes to be tested. The present invention can realize high-sensitivity detection through nanoscale excitation wavelength shift changes, and can perform identification and detection outside cells. The whispering gallery mode makes the fluorescence resonate in the cavity to generate laser light, which prolongs the tracking detection time and realizes the tracking and identification function according to the enhanced detection signal.

Specifically include the following steps:

Step 1: The microcavity used in this experiment can be a solid microcavity such as a microsphere cavity, a microring cavity, a microdisk cavity, etc. The fabrication material of the microcavity can be a silicon compound with a small propagation loss, an amorphous glass material, and a polymer. materials, etc., microcavities are prepared by high-temperature melting and sol-gel methods. In the microcavity, one of the fluorescent substances such as dyes, quantum dots, fluorescent proteins, etc. can be selected to be doped. The laser with the wavelength that meets the corresponding conditions is coupled into the microcavity to form resonance, and the laser is excited when the threshold condition is reached.

Step 2: Functionalize the surface of the fluorescent microcavity, purchase carboxyl-modified or amino-modified fluorescent microcavities, and then combine with amino- or carboxyl-modified aptamers or specific antibody proteins through covalent coupling between amino and carboxyl groups, Thus, the fluorescent microcavity can specifically identify the exosomes of different cells to be detected.

Step 3: Covalently couple the carboxyl- or amino-modified fluorescent microcavity with an amino- or carboxyl-modified aptamer or specific antibody protein that can specifically bind to the corresponding exosomes. After 2-4 hours, the carboxyl- or amino-modified fluorescent microcavity specifically recognizes the amino- or carboxyl-modified aptamer, and the amino-carboxyl groups are linked together by dehydration condensation reaction; connect the connected microcavity-aptamer to the corresponding cell The extracted exosomes were incubated together for 0.5 hours, and the aptamer specifically recognized and bound to the surface of the corresponding exosomes. Alternatively, a purchased fluorescent microcavity modified with an antibody that specifically recognizes exosome surface antigens was mixed and incubated with exosomes for 0.5 hours. After the successful connection was verified by confocal detection, the microspheres were then irradiated with a laser. The excited fluorescence is coupled into the microsphere to generate laser in the whispering gallery mode, and the external spectrometer receives the spectral signal. In this experiment, the spectral signal of the fluorescent microcavity-aptamer without exosomes is used as a control.

The main application of this experiment is the mode-shift sensing mechanism, that is, the resonant wavelength of the whispering gallery mode changes with the change of the environment, and the larger the resonant wavelength changes, the higher the concentration of the detected molecules; at the same time, different resonant wavelengths correspond to different test substance. The laser is coupled into the interior of the microcavity, and total reflection occurs mainly in the cavity, but there is still an optical field in the second medium (air or liquid on the outer surface of the microcavity), that is, the evanescent field, with a distribution range of about a few hundred on the surface of the microsphere. nanometer, so when exosomes bind to the surface of the microcavity, it will change the evanescent field, resulting in frequency drift, and because of the high quality factor Q and narrow linewidth characteristics of the microcavity, the spectral drift signal is amplified, so it can pass the spectrometer. Observe the frequency shift to detect exosomes secreted by specific cells. Different exosomes have different perturbation results to the evanescent field, and the corresponding frequency shifts are also different, so as to distinguish different exosomes.

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

1. The detection method is simple, the cost is low, and the sensitivity is high, and some substances in the cell can be detected outside the cell. In this experiment, the laser was used to detect exosomes based on the whispering gallery mode. The polystyrene fluorescent microspheres used were 15 μm in diameter, with high quality factor Q and extremely narrow line width, so the sensitivity was high and the production cost was low; Exosomes not only contain a variety of proteins that can be used as markers, but also exosomes secreted by cells also contain a large number of small nucleic acid molecules. The high sensitivity of this method enables detection of low concentrations.

2. To achieve extracellular detection. The microsphere acts as a resonant cavity in the cell, and the laser output is generated by the whispering gallery mode, acting as a cellular laser. A large number of studies have shown that exosomes can participate in the occurrence, growth, and growth of normal cells and cancer cells by transferring cancer-causing proteins and nucleic acids. Progress and transfer, then the cell laser can track the movement of exosomes, detect at different locations, and have little effect on cells, making it more practical.

3. The detection based on the whispering gallery mode makes up for the self-absorption and bleaching of the fluorescent signal, so that the fluorescence forms a resonance in the cavity to excite the laser, and the signal intensity is enhanced to facilitate detection, prolong the observation and detection time, and increase the reliability and reliability of detection. stability.

Description of drawings:

1 is the structure diagram of the experimental device of the present invention: 1, 532nm pulsed laser; 2, attenuator; 3, silver mirror; 4, CCD camera; 5, focusing lens; 6, dichroic mirror; 7, 40 times microscope Objective lens; 8. Sample; 9. 60x microscope objective lens; 10. Filter; 11. Focusing lens; 12. Transmission fiber and probe; 13. Spectrometer.

Detailed ways

The present invention will be further described below in conjunction with the implementation examples, but the present invention is not limited to the following implementation examples. Because exosomes contain the same nucleic acid and protein molecules as cells, we can detect the corresponding microRNAs and proteins by detecting different types of cells, including various normal cells and cancer cells.

Example 1 Detection of breast cell SK-BR3 exosomes using active microsphere chambers

In this experiment, polystyrene fluorescent microspheres modified with carboxyl groups were selected. The diameter of the microspheres was 15 μm, the refractive index was n=1.59, and the microspheres had a high Q value and a small mode volume.

The laser light source used in this experiment is a 532nm pulsed laser. Different lasers use different excitation wavelengths of microspheres. The detected substances are exosomes secreted by breast cells SK-BR3. The exosomes were extracted, and then the carboxyl-modified polystyrene fluorescent microspheres with a concentration of 0.1 kg/L were suspended in MES buffer, and 1-ethyl-3-(dimethylaminopropyl) carbodiimide solution was added. and N-hydroxysuccinimide solution, mix the amino-modified specific sequence aptamer and carboxyl-modified fluorescent microspheres at a concentration of 0.5 μmol/l and stir at room temperature for 2-4 hours. After centrifugation and washing, the aptamer will be successfully connected. The fluorescent microspheres were mixed with the extracted exosomes and incubated at room temperature for 0.5 hours. After washing, confocal was used to check whether the connection was successful, and then the droplets were taken out with a pipette and placed in Poly-D-lysine. On the acid-cleaned cover glass, finally place the slide sample on the three-dimensional adjustment frame, adjust the adjustment frame, make the laser freely couple into the microsphere, increase the laser power to the laser excitation threshold of the microsphere, and observe on the spectrometer To the laser, the presence of such mammary cell exosomes was determined by spectral shift compared to the excitation spectrum of the microspheres bound only with aptamers.

The specific experimental steps are as follows:

(1) Measure the spectrum of carboxyl-modified polystyrene fluorescent microspheres. Take out the carboxyl-modified polystyrene fluorescent microspheres with a concentration of 0.1kg/L, and suspend them in PBS buffer with pH=7.4 and shake well. The volume ratio of microspheres to PBS buffer is 1:10. The mixed solution was dropped on the coverslip cleaned by Poly-D-lysine, that is, poly-lysine, to prevent the microspheres from moving. The carboxyl-modified microspheres were pumped by the device in the figure, and the laser output was measured. The central peak of the laser is at 610 nm, and the spectral output is recorded as a reference to calculate the frequency shift value for subsequent experiments.

(2) Measure the spectrum of the linked aptamer-microsphere. First, add the following substances to the centrifuge tube: 0.5 μmol/l amino-modified aptamer, 0.1 kg/L carboxyl-modified microspheres with a diameter of 15 μm, 0.1 mol/l 1-ethyl-3-(dimethylformaldehyde) Aminopropyl) carbodiimide solution is abbreviated as EDC, 0.1mol/L N-hydroxysuccinimide solution is abbreviated as NHS, pH=5.5 MES buffer, the volume ratio of the above substances is: aptamer: microsphere : 1-ethyl-3-(dimethylaminopropyl) carbodiimide solution: 0.1 mol/L N-hydroxysuccinimide solution: MES=1:1:5:5:10. Rotate and incubate for 2-4 hours at room temperature; then need to centrifuge for washing, centrifuge for 5 minutes, remove the supernatant, and resuspend in PBS buffer pH=7.4; then centrifuge for 5 minutes, remove the supernatant, and finally The particles were resuspended in PBS buffer to obtain a suspension. Similarly, drop the connected aptamer-microspheres on the treated coverslip with a pipette, use the device diagram to pump the aptamer-microspheres with a 532nm laser, and measure the laser spectrum of the aptamer-connected microspheres , the central peak of the laser excited at the current concentration is 609.3 nm, and it is found that the central peak of the laser has shifted. Change the concentration of the added aptamer, and perform excitation again with other experimental conditions unchanged to compare with the spectrum in step (1), and draw a frequency shift-concentration standard curve according to the frequency shift of the spectrum line. Change the concentration of the amino-modified aptamer with a specific sequence, and measure the laser spectrum at the corresponding concentration. According to the principle of the whispering gallery detection substance, when the frequency shift is the largest, it indicates that the aptamer has the highest binding efficiency, so it can be calculated and determined as the best Aptamer ligation efficiency.

(3) Determination of the presence of specific exosomes. The mammary gland cells were first cultured with exosome-free serum, and then the exosomes secreted by the cells were extracted with an exosome kit; the connected aptamer-microspheres were incubated with the extracted exosomes for 0.5 hours , drop it on the cover glass cleaned by Poly-D-lysine (poly-lysine) with a pipette, record the laser excitation spectrum under 532nm laser pumping using the device diagram, the current central peak of the laser is 607.6nm , compared with the reference spectrum in (2), it was found that the central peak of the laser was shifted, and the presence of the exosome was determined according to the measured frequency shift.

(4) As a result of the experiment, the detection of intracellular substances can be quickly and sensitively realized outside the cell.

Example 2 Using the active microsphere cavity to detect the prostate-specific antigen (PSA) of biological macromolecules on exosomes secreted by prostate cells

Currently, the detection of PSA is mainly invasive, including by digital rectal examination, transrectal ultrasound-guided biopsy, etc. The use of active cavity to detect exosomes has the advantages of non-invasiveness, and it is more convenient and fast to measure PSA. The research content of the present invention is to use specific aptamer-modified active microspheres to detect exosomes secreted by prostate cells. PSA was detected.

The specific experimental steps are as follows:

(1) First purchase carboxyl-modified fluorescent microspheres;

(2) The aptamer to be modified, the aptamer sequence is 5'-TTATTAAAGCTCGCCATCAAA TAGC-3'-NH2, and the carboxyl group-modified microsphere, according to the formula S=(6/ρSd)·C to calculate the required aptamer concentration , where S is the amount of aptamer required for monolayer modification (mg aptamer/g microsphere), ρS is the density of the microsphere (g/cm ³ ), d is the diameter of the microsphere, and C is the capacity of the microsphere surface .

(3) The carboxyl group-modified microspheres were placed in MES solution with pH=5.5, and then EDC and NHS were added to activate the carboxyl group in a suitable environment. Then, the aptamer was added, and the fluorescent microspheres successfully connected to the aptamer were obtained by vortexing and centrifugal washing.

(4) Extract and purify exosomes, first use bovine serum containing normal exosomes to culture the prostate cells, the growth state is good, and when the number of cells reaches 70%-80% within the field of view, use bovine serum without exosomes to carry out Subculture, the growth state is good, and when the number of cells reaches 70%-80% within the field of view, use a kit for extraction and purification.

(5) The fluorescent microspheres successfully connected to the aptamer and the extracted and purified exosomes were mixed together and incubated for 0.5 hours, and the successful connection was verified by confocal.

(6) Under the pumping of a 532nm pulsed laser, record the laser spectrum, and compare with the spectra of the carboxyl-modified microspheres and the aptamer-connected microspheres, and found that the carboxyl-modified microspheres and the aptamer-connected microspheres were successfully The laser spectra of the spheres and the microspheres successfully connected to exosomes shifted sequentially. When the concentrations of exosomes in the connected samples were different, the central peak of the laser spectrum was compared with the central peak of the laser spectrum of the successfully connected aptamer. , the displacement size of the movement is also different. From this, the frequency shift of the spectrum can be used to observe the presence of PAS, as well as the change in concentration.

(7) Experimental results, using this method to detect can achieve a fast and efficient detection effect.

Example 3 Detection of Glypican-1 in exosomes secreted by pancreatic ductal cells using the active microsphere cavity

Currently, the relevant detection methods for pancreatic ductal cells are mainly expensive imaging studies, such as computed tomography (CT), magnetic resonance imaging (MRI), and endoscopic ultrasound. Glypican-1 has recently been identified as a biomarker for exosomes. The present invention is based on the exosome marker glypican-1, uses carboxyl-modified fluorescent microspheres and amino-modified GPC-1-recognizing antibodies to recognize and bind through the covalent coupling of amino carboxyl groups, and then mixes with exosomes, which will be combined with exosomes. The marker GPC-1 on the surface of exosomes is specifically recognized and bound by antigen and antibody, so that the emission spectrum of the laser at this time is shifted compared with the laser emission spectrum of the carboxyl-modified microcavity that does not recognize the exosome binding. The greater the concentration of exosomes, the greater the shift will be, so the concentration of the substance can be detected by the magnitude of the spectral shift.

The specific experimental steps are as follows:

(1) First buy fluorescent microspheres with carboxyl group modification:

a) adding pH=5.5 MES solution, 1-ethyl-3-(dimethylaminopropyl) carbodiimide) solution and N-hydroxysuccinimide solution to activate carboxyl-modified fluorescent microspheres;

b) Carboxyl-modified microspheres bind to amino-modified GPC-1-recognizing antibodies.

(2) The pancreatic cells U87-MG were cultured, and the kit was used to extract exosomes when the growth state was good.

(3) The fluorescent microspheres linked to specific antibodies were incubated with exosomes for 0.5 hours, and after washing, the carboxyl-modified microspheres, the microspheres linked to specific antibodies, and different concentrations of The exosome-bound microspheres were pumped separately, and three laser spectrum data were obtained in turn. By comparing the central peaks of the three-excited laser spectra, it was found that the wavelength of the central laser peak would shift in sequence, and the aptamer was changed during sample preparation. The concentration and the concentration of extracted exosomes, the magnitude of the shift of the laser center peak will also change accordingly.

(4) Identify the presence and concentration of GPC-1 according to the output spectral frequency shift map.

The core part of the present invention lies in the laser excitation of the whispering gallery mode to achieve extracellular laser output. When the modified active fluorescent microspheres are specifically combined with antibodies or aptamers, spectral frequency shift occurs, thereby achieving high sensitivity. Extracellular material detection.

Claims (7)

1. A method for detecting exosomes based on whispering gallery mode optical microcavities is characterized by comprising the following specific steps:

step 1: a detection device is set up, a nano pulse laser is fixed on an optical platform, so that a transmitted beam is emitted horizontally, the beam passes through an attenuation sheet and is used for adjusting the light intensity emitted to the surface of a sample, then passes through a silver mirror, the beam path is changed into a right angle with the original direction, the purpose is to increase the setting up of a rear light path in the setting up space, a dichroic mirror is added along the beam direction, so that the reflected beam is parallel to the original beam, a 40-time microscope objective is fixed along the reflected beam direction, the beam is collimated to pass through, then a 60-time microscope objective is fixed behind the 40-time objective along the beam direction, the distance between the two microscope objectives is the sum of the focal distances of the two objectives, namely the focal points are overlapped, in the experimental process, a sample is placed at the focal point overlapped part of the two objectives and is used for exciting a microcavity, then a filter is fixed, a focusing lens is fixed behind the filter plate and used for collecting fluorescence and laser and transmitting the fluorescence and the laser to a receiver optical fiber probe of a spectrometer behind the focusing lens more efficiently; a focusing lens is fixed behind the focusing lens along the direction of the transmitted light beam and is used for displaying an image on a CCD camera behind and observing the condition that the light beam acts on the surface of the microcavity;

step 2: the laser is incident into the echo wall micro-cavity with the size of several to dozens of microns, and is totally reflected inside the micro-cavity doped with the fluorescent material, when the total reflection optical path is integral multiple of the wavelength, the total reflection optical path forms a resonant cavity, and when the light meets the condition that the gain is greater than the loss, a whispering gallery mode micro-cavity for detection is formed;

step 2: the method comprises the following steps of treating the echo wall microcavity of which the surface is modified with carboxyl or amino and is doped with fluorescent substances, wherein the step is mainly to mix and react an aptamer modified with amino or carboxyl or specific antibody protein modified with amino or carboxyl with the corresponding echo wall microcavity modified with surface carboxyl or amino, and the target detection object and a modifier on the surface of the microcavity are identified and combined through automatic covalent coupling of amino and carboxyl;

and step 3: incubating the echo wall microcavity connected with the aptamer or the specific antibody protein with the exosome, and specifically identifying and combining the aptamer or the specific antibody protein successfully connected with the microcavity surface and the specific protein on the corresponding exosome surface by base complementary pairing;

and 4, irradiating the microcavity with a laser, centrifuging after the exosome is successfully connected with the microcavity, washing off the exosome and the microcavity which are not successfully connected, suspending the exosome and the microcavity in PBS (phosphate buffer solution) with the pH value of 7.4, dripping the microcavity successfully connected with the exosome on a glass slide, putting the glass slide in a light path, focusing and coupling laser beams into the microcavity by adjusting the position of the glass slide, firstly, adjusting the intensity of the light beams by using an attenuation sheet to enable the light beams to reach an ASE (amplified spontaneous emission) signal for exciting fluorescence at an excitation threshold, then, rotating the attenuation sheet to enable the light power emitted to the surface of the microcavity to increase the light intensity by increasing the light power of 5 muW each time, enabling the light beams to emit laser in a whispering gallery mode, externally connecting a spectrometer and a CCD (charge coupled device) and combining the microcavity and the exosome.

2. The detection method according to claim 1, wherein the fluorescent substance is a fluorescent dye, a quantum dot, or a fluorescent protein.

3. The detection method as claimed in claim 1, wherein the surface treatment comprises specific antibody protein modification or related detection aptamer modification.

4. The detection method of claim 1, wherein the echo wall microcavity material is selected from the group consisting of: the surface is modified with carboxyl or amino micro-cavity.

5. The detection method according to claim 1, wherein the selection of the laser light source: the nano pulse laser detects the cell secretion exosome produced by various cells.

6. The detection method according to claim 1, wherein the exosome molecules in the culture solution are detected first, and purchased microcavities with the surface modified by carboxyl or amino groups and the mass concentration of 0.1kg/L are arranged in a proportion of 1: adding MES buffer solution according to the volume ratio of 10, centrifuging, shaking, washing, then resuspending in the MES buffer solution, wherein the pH value of the MES buffer solution is 5.5, adding 1-ethyl-3- (dimethylaminopropyl) carbodiimide solution with the concentration of 0.1mol/L according to the volume ratio of 1:5 of the microcavity and 1-ethyl-3- (dimethylaminopropyl) carbodiimide solution, then adding N-hydroxysuccinimide solution with the concentration of 0.1mol/L according to the volume ratio of 1:5 of the microcavity and the N-hydroxysuccinimide solution, so that the carboxyl group on the surface modification of the microcavity promotes the recognition and the combination with the amino group modified aptamer and the amino group modified specific antibody protein, or the carboxyl group on one end of the carboxyl group modified aptamer and the carboxyl group modified specific antibody protein promotes the recognition and the combination with the amino group modified microcavity, and mixing uniformly, adding amino-group or carboxyl-group modified aptamer or specific antibody protein into a microcavity the surface of which is modified by carboxyl or amino-group and an amino-group or carboxyl-group modified aptamer or specific antibody protein according to the volume ratio of 1:1, performing vortex oscillation for 3 hours, performing dehydration condensation on the amino-group and the carboxyl group to realize connection of the aptamer or specific antibody protein and the microcavity, centrifuging for 5min, washing off redundant aptamer, and resuspending the centrifuged sample in PBS buffer according to the volume ratio of the microcavity to the PBS buffer of 1: 8; then uniformly mixing a connecting body microcavity-aptamer or microcavity-specific antibody protein re-suspended in a PBS buffer solution with exosome molecules with the concentration of 0.5 mu mol/l according to the volume ratio of 1:1, realizing specific recognition and combination of the microcavity and the exosome molecules through the specific combination characteristic of the protein, centrifuging for 5min through a centrifuge after mixing and incubation, and re-adding the PBS buffer solution with the pH value of 7.4; then placing the PBS buffer solution containing the mixture microcavity, the aptamer or the specific antibody protein, the exosome and the microcavity-aptamer-exosome in a centrifuge tube, centrifuging for 5min, and then resuspending in the PBS buffer solution for storage;

taking out the culture solution successfully connected by using a pipette and dripping the culture solution on a cover glass to manufacture a glass slide; finally, laser is focused and coupled into the micro-cavity, a CCD camera is used for imaging to observe and adjust light beams to focus the light beams on a single micro-cavity, the spectral positions before and after the connection with the exosome are compared and analyzed through spectral recording on a spectrometer, and the existence of the exosome is judged by finding the movement of the front and back comparison spectra; and comparing and analyzing the spectral shift generated by the exosomes secreted by different cells to judge the types of the cells.

7. The method for detecting exosomes according to claim 1, wherein the specific experimental steps for detecting exosomes secreted by cells are as follows:

(1) firstly, connecting a purchased amino or carboxyl modified aptamer with a purchased carboxyl or amino modified fluorescent microsphere, and calculating the quantity concentration and volume of substances of the amino or carboxyl modified aptamer corresponding to the connection of the carboxyl or amino modified microsphere according to the principle that the amino and the carboxyl are connected in a dehydration condensation manner according to the quantity ratio of 1: 1;

(2) the purchased fluorescent microspheres with the surfaces modified by carboxyl or amino are mixed according to the ratio of 1: adding MES buffer solution according to the volume ratio of 10, centrifuging by a centrifuge, shaking for 5 minutes, washing, and then resuspending in the MES buffer solution, wherein the pH value of the MES buffer solution is 5.5; the following were then added to the centrifuge tube: 0.5 mu mol/L of aptamer with a base sequence capable of identifying secretion exosomes of cells to be detected, wherein one end of the aptamer is modified with amino or carboxyl, 0.1mol/L of 1-ethyl-3- (dimethylaminopropyl) carbodiimide solution and 0.1mol/L of N-hydroxysuccinimide solution; the volume ratio of the fluorescent microsphere modified by carboxyl or amino is as follows: fluorescent microsphere, aptamer, 1-ethyl-3- (dimethylaminopropyl) carbodiimide solution and N-hydroxysuccinimide solution, wherein the aptamer is 1:1:5

(3) Rotationally stirring and incubating for 3 hours at room temperature, then centrifugally washing, centrifuging for 5 minutes, removing supernatant, washing out 1-ethyl-3- (dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and unsuccessfully connected aptamer and microsphere, then adding PBS buffer solution with the pH value of 7.4, and suspending the fluorescent microcavity successfully connected with the specific base sequence in the PBS buffer solution;

(4) putting the suspension into a centrifuge tube, centrifuging for 5min, removing supernatant, adding PBS buffer solution to resuspend the exosome-aptamer-microsphere connecting body in the PBS buffer solution for storage;

(5) culturing the mammary cells for 48 hours by using the exosome-free serum, and extracting exosomes by using an exosome extraction kit;

(6) adding a buffer solution with the pH value of 7.4 into the extracted exosome to dilute the exosome into different concentrations;

(7) exosomes of different concentrations were mixed with sample solutions of exosome-aptamer-microsphere linkers suspended in PBS buffer at 1:1, evenly mixing and incubating for 0.5 hour;

(8) centrifuging the sample solution after the seventh step reaction in a centrifuge for 5min, washing twice, and then resuspending in a buffer solution of PBS (phosphate buffer solution) with the pH value of 7.4;

dripping the successfully connected exosome-aptamer-fluorescent microspheres on a cover glass cleaned by using Poly-D-lysine (polylysine) by using a pipette gun, exciting the exosome-aptamer-microspheres by using a 532nm pulse laser, measuring and recording an excitation spectrum of the exosome-aptamer-microspheres, comparing the excitation spectrum with an excitation spectrogram of the aptamer-fluorescent microspheres, measuring the frequency shift of a spectral line, and drawing a frequency shift-concentration standard curve;

because need use the spectrum appearance to detect the spectrum, so need go on under the shading condition, in order to guarantee the life of the optical system who builds, need guarantee indoor cleanness simultaneously.

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