Real-time enrichment and detection method for extracellular vesicles

Abstract

The invention relates to a real-time enrichment and detection method for extracellular vesicles. Belongs to the technical field of biological detection. The preparation method comprises the following steps: firstly, preparing a silicon dioxide colloidal crystal (SCC) thin film by adopting a vertical evaporation method; then modifying distearoyl phosphatidyl ethanolamine-polyethylene glycol-carboxyl (DSPE) on the silicon dioxide colloidal crystal film, and separating and enriching the extracellular vesicles in the biological liquid by utilizing the affinity characteristic of the DSPE and a phospholipid bilayer; then constructing a reflection interference spectrum system, and detecting the extracellular vesicles in real time by utilizing optical thickness change caused in the process of separating the extracellular vesicles; according to the method, the extracellular vesicles in various biological liquids can be rapidly and efficiently separated, and real-time concentration and membrane protein expression quantity analysis can be carried out on the separated extracellular vesicles; the invention provides a rapid and low-cost integrated multifunctional analysis method for separation of extracellular vesicles of various body fluids.

Description

Technical Field

[0001] The invention relates to a real-time enrichment and detection method for extracellular vesicles, belonging to the technical field of biological detection. Background Art

[0002] Extracellular vesicles (EVs) are nanoscale particles derived from cells and have a lipid bilayer structure. They are rich in a variety of bioinformatic molecules, including proteins, mRNA, non-coding RNA, DNA, lipids and other components. In recent years, the scientific community has shown increasing interest in the study of EVs because they have shown significant therapeutic potential in multiple therapeutic areas such as tissue repair and regeneration, infectious diseases, autoimmune diseases and cancer. EVs are widely distributed in various body fluids such as blood, urine, saliva, cerebrospinal fluid and peritoneal fluid. As a direct reflection of the state of parent cells, their stable cell structure has brought unprecedented convenience and possibilities to clinical testing, indicating that EVs are expected to become an important part of liquid biopsy technology. Protein molecules located on the surface or inside EVs provide rich, stable, sensitive and unique biological information. These proteins have been successfully developed into specific diagnostic and prognostic assessment factors for a variety of diseases such as inflammation, neurodegenerative diseases and cancer. More importantly, when using membrane proteins as markers for detection, there is no need to destroy the physical structure of EVs or further cut them, thereby maintaining the physiological activity and integrity of EVs and their contents, which is undoubtedly a major advantage in the field of detection.

[0003] The small size and low density of EVs make it challenging to specifically separate them from a wide range of cell fragments and complex components of biological fluids. Ultracentrifugation (UC) is currently the most commonly used method for isolating EVs in the laboratory and is also the gold standard method; however, ultracentrifugation requires expensive ultracentrifuges, the extraction process requires multiple tedious purification steps, the processing time is long, and the separation efficiency is generally less than 30%. In addition, there are methods such as immunoprecipitation, precipitation polymerization and ultrafiltration, but the separation efficiency and purity of these methods cannot meet the needs of scientific research. Moreover, these methods will destroy the structure and function of extracellular vesicles, and the separated extracellular vesicles are prone to aggregate into clusters, which is not conducive to downstream analysis. At the same time, at this stage, the enrichment and detection of EVs are carried out separately. When detecting, EVs often need to be separately packaged and tested, resulting in the loss of EVs, resulting in the detection value being lower than the enrichment value.

[0004] Therefore, there is an urgent need to develop a method that is simple, economical, and can quickly separate and enrich EVs from complex samples and detect them in real time. Summary of the invention

[0005] In view of some deficiencies in the prior art, the purpose of the present invention is to provide a method for real-time enrichment and detection of extracellular vesicles. The method of the present invention can be used to enrich extracellular vesicles and detect the concentration of extracellular vesicles in real time; further, the method of the present invention can be used to analyze the expression level of corresponding extracellular vesicle membrane proteins and the kinetics of their binding and dissociation with antibodies.

[0006] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0007] The present invention first provides a method for real-time enrichment and detection of extracellular vesicles, the method comprising the following steps:

[0008] S1. Preparation of silica colloidal crystal film (SCC film) by vertical deposition method;

[0009] The method for preparing a silica colloidal crystal film by vertical deposition method comprises:

[0010] The silica microspheres were centrifuged and then repeatedly washed with anhydrous ethanol and centrifuged; the glass slides were immersed in piranha solution and then rinsed with ultrapure water; the treated slides were placed vertically in a silica ethanol suspension (1%), and after ethanol evaporation for 7 days, a large area of ​​silica colloidal crystal film was formed on the glass surface.

[0011] Furthermore, in step (3), the evaporation environment temperature is 25±5° C. and the humidity is 10±5%.

[0012] S2. Preparation of DSPE-modified silica colloidal crystal films;

[0013] The preparation method of the DSPE-modified silica colloidal crystal film comprises:

[0014] The SCC film is immersed in an ethanol solution of aminopropyltriethoxysilane to obtain an amino SCC film; DSPE is dissolved in an ethanol solution to obtain a solution 1, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are dissolved in a Tris-HCl buffer to obtain a solution 2, and the solution 1 and the solution 2 are mixed and the pH value is adjusted to obtain a mixed solution; the amino SCC film is reacted with the mixed solution, and then washed with ultrapure water to obtain a DSPE-modified silica colloidal crystal film, which is stored in ultrapure water for later use;

[0015] Further, the concentration of the aminopropyltriethoxysilane ethanol solution is 0.1%-2% (volume / volume), and the SCC film is immersed in the aminopropyltriethoxysilane ethanol solution for 1-48 hours;

[0016] Furthermore, the dosage of the DSPE and ethanol solution is 1-100 mg: 1 mL, and the dosage of the N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and Tris-HCl buffer is: 20-200 mg: 50-400 mg: 4 mL.

[0017] Furthermore, after the solution 1 and the solution 2 are mixed, the pH value is adjusted to 7.5; and the reaction time is 4-12 hours.

[0018] S3. Set up OPLI (ordered porous layer interferometry) system:

[0019] A reaction cell was assembled with DSPE-modified silica colloidal crystal film as the substrate, and an OPLI (ordered porous layer interferometry) system was formed with an inverted microscope and a fiber optic spectrometer.

[0020] Furthermore, the establishment of the OPLI system includes the following steps:

[0021] Drill a hole on the silicone pad and cover it on a glass slide of silica colloidal crystal film modified with DSPE to form a reaction pool; press the upper end with another glass slide to form a closed reaction pool; fix the reaction pool on an inverted microscope and adjust the focus to the silica colloidal crystal layer; couple the optical fiber through a spectroscope and connect it to a spectrometer; connect the catheter to the reaction pool and connect the catheter to a peristaltic pump so that the reaction liquid can flow into the hole in the middle of the silicone pad, repeat the operation to connect another catheter so that the reacted liquid can flow out.

[0022] S4. Isolation and enrichment of extracellular vesicles:

[0023] PBS solution was introduced into the reaction pool of the OPLI system. After the baseline was stabilized, the sample solution of the extracellular vesicles to be separated was recirculated. When the baseline displayed by the spectrometer was stable, the spectral line was fitted for the selected reflection interference spectrum band. The optical thickness of the silica colloidal crystal film was calculated by extreme value fitting to be 1, thus completing the separation and enrichment of the extracellular vesicles.

[0024] Furthermore, the sample solution for separating extracellular vesicles includes cell culture fluid, urine, milk, cerebrospinal fluid or blood.

[0025] S5. Dissociation of enriched extracellular vesicles:

[0026] After the enrichment is completed, detergent is introduced to wash away impurities adsorbed on the SCC film; finally, elution solution is introduced to dissociate the extracellular vesicles enriched in the SCC film.

[0027] Furthermore, the detergent includes Triton or ethylphenyl polyethylene glycol, and the elution solution includes triethanolamine.

[0028] S6. Establish a linear relationship and analyze the concentration of extracellular vesicles:

[0029] The sample solution of the extracellular vesicles to be separated at different dilution ratios is circulated, and steps S4 and S5 are repeated. A linear relationship is established with the optical thickness 1 as the ordinate and the dilution ratio as the abscissa to analyze the extracellular vesicle concentration.

[0030] S7. Repeat steps S1-S5, except that in step S5, the elution solution is not passed, but the extracellular vesicle membrane protein antibody and PBS solution are passed; the selected reflection interference spectrum band is fitted with spectra, and the optical thickness 2 and optical thickness 3 of the silica colloidal crystal film are calculated by extreme value fitting method, and the binding and dissociation curves of the membrane protein and the antibody are obtained, and the expression level of the corresponding extracellular vesicle membrane protein and the kinetics of its binding and dissociation with the antibody are analyzed.

[0031] Furthermore, the optical thickness in step S4 and step S7 is calculated according to the formula Fitting is performed, wherein λ is the real-time wavelength of the interference peak in the range of 430-1100nm, that is, the selected reflection interference spectrum band, k′ is the relative order, k′=0, 1, 2, 3, 4···, nd is the optical thickness; the extracellular vesicle membrane protein antibody includes antibodies corresponding to membrane proteins CD9, CD63 or CD81.

[0032] The working principle of the technical solution of the present invention is described as follows:

[0033] The DSPE on the modified silica colloidal crystal film will specifically bind to the phospholipid bilayer of EVs, thereby separating the EVs in the sample. The OPLI system is based on the reflection interference phenomenon caused by the reflection of white light at the upper and lower boundaries of the SCC film layer. The wavelength of the interference peak is converted into a change in optical thickness (ΔOT) for observation by the extreme value tracking method. When ΔOT changes from rising to stable, it indicates that the separation of EVs is complete, and then the elution solution is introduced to elute the EVs that have been fixed on the SCC film to achieve separation. Directly introducing the corresponding antibody without the elution process will bind to the surface membrane protein of the EVs fixed on the SCC film. The first-order kinetic model is fitted by the real-time optical thickness change curve to calculate the information such as the binding rate constant, dissociation rate constant and equilibrium constant of the antibody and membrane protein, thereby verifying that the method provided by the present invention can analyze the binding and dissociation constants of EV membrane proteins and antibodies or drugs, and can further be used for drug development or target protein screening.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The method described in the present invention uses a silica colloidal crystal film modified with DSPE to enrich EVs. The entire process does not require the use of large instruments and is simple to operate and time-saving. The entire EV separation process can be completed within 1 hour.

[0036] (2) The method described in the present invention uses the silica colloidal crystal film modified with DSPE as the substrate to assemble into a reaction pool, and forms an OPLI system with an inverted microscope and a fiber optic spectrometer, which can obtain a real-time curve of the combination of EVs and the silica colloidal crystal film. The real-time curve can observe the start and end of the EVs capture process. At the same time, the capture process can be recorded in real time through ΔOT, and the EVs separation process can be visually observed. The scheme of the present invention can calculate the specific concentration of EVs while separating EVs, predict the quality of EVs in the sample, and achieve simultaneous separation and detection; there is no need to separately subpackage and monitor the samples again, which reduces sample loss, simplifies the operation process, and realizes the separation and concentration detection of EVs in one step. The efficiency and concentration of separation and capture of EVs are very high.

[0037] (3) The method described in the present invention can also fix the separated EVs on the silica colloidal crystal film, analyze the expression of EV membrane proteins from different sources, and perform antibody screening and kinetic detection of their surface membrane proteins; at the same time, the expression of EV surface membrane protein markers of the corresponding disease can be analyzed to realize the liquid biopsy function; in view of the porous and pore size characteristics of the silica colloidal crystal film, the signal collection area will not be affected by micron-level cells. At the same time, the OPLI system uses an inverted microscope with an integrated optical path system, and the flow of samples above the film will not interfere with the system monitoring. Therefore, it can be applied to more and more complex clinical samples, such as cerebrospinal fluid, emulsion and blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The scanning electron microscope images of the silica colloidal crystal film prepared in Example 1, wherein Figure A is a front view of the silica colloidal crystal film, with a scale of 1 μm; Figure B is a cross-sectional view of the silica colloidal crystal film, with a scale of 200 nm.

[0039] Figure 2 This is the reflection interference spectrum of the silica colloidal crystal film prepared in Example 1.

[0040] Figure 3 This is the front and rear reflection interference spectrum of the silica colloidal crystal film modified with DSPE prepared in Example 1.

[0041] Figure 4 This is a real-time optical thickness change diagram during the separation process of extracellular vesicles in the cell culture supernatant in Example 1.

[0042] Figure 5 This is a graph showing the results of a CD9 protein blotting experiment of EVs in the cell culture supernatant completely separated and eluted by the method of the present invention and the UC method.

[0043] Figure 6 This is a transmission electron microscopy photograph of EVs after complete separation and elution in Example 1. The scale bar is 200 nm.

[0044] Figure 7 This is the real-time optical density change during the separation of extracellular vesicles in urine in Example 2.

[0045] Figure 8 This is a graph showing the results of a CD9 protein blotting experiment of EVs in urine eluted by the method of the present invention and the UC method.

[0046] Fig. 9 This is a transmission electron microscopy photograph of EVs after complete separation and elution in Example 2. The scale bar is 200 nm.

[0047] Fig.10 This is a real-time optical thickness change diagram during the separation process of extracellular vesicles in the supernatant of MSC culture fluid diluted at different ratios in Example 3.

[0048] Fig.11 It is a linear relationship diagram in Example 3 with different dilution ratios as the horizontal axis and the change in optical thickness as the vertical axis.

[0049] Fig.12 This is the real-time curve of optical thickness of real-time EVs separation in the supernatant of MSC culture fluid in Example 4 and the real-time curve of CD9 antibody binding to membrane protein.

[0050] Fig.13 In Example 4, it is a real-time curve graph of the optical thickness changes of the binding and dissociation of EVs membrane proteins with CD9, CD63 and CD81 antibodies in the supernatant of MSC culture fluid.

[0051] Fig.14 This is a graph showing the change in optical thickness caused by the circulation of the αvβ3 antibody in Example 5 and its binding to the fixed EVs in the urine sample of a prostate cancer patient.

[0052] Fig.15 This is a graph showing the change in optical thickness caused by the circulatory introduction of the Trop-2 antibody in Example 5 and its binding to the fixed EVs in the urine sample of a prostate cancer patient.

[0053] Fig.16 This is a diagram of the change in optical thickness caused by circulating the tumor marker antibody αvβ3 and Trop-2 solution after EVs were fixed in the urine samples of prostate cancer patients and healthy people in Example 5. DETAILED DESCRIPTION

[0054] In order to enable those skilled in the art to better understand the technical solution of the present invention, the preferred embodiments of the present invention are described in detail below, but the following embodiments do not limit the protection scope of the present invention.

[0055] In the embodiments of the present invention, those that are not described in detail are all completed by conventional experimental methods. Those processes involved in the embodiments that are not described in detail are all understandable and easily implementable by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore are not described in detail.

[0056] In the following examples, the diameter of the aqueous dispersed silica microspheres is 190 nm, purchased from Nissan Chemical Co., Ltd. of Japan; DSPE-PEG2000-COOH is purchased from Xi'an Ruixi Biological Co., Ltd.; the fiber optic spectrometer is a Fuxiang PG2000 PRO spectrometer; unless otherwise specified, the drugs and reagents involved are common commercial products.

[0057] The piranha solution described in the examples is prepared by conventional methods.

[0058] Example 1: Isolation and enrichment of EVs in MSC (mesenchymal stem cell) culture supernatant

[0059] S1. Preparation of silica colloidal crystal film (SCC film)

[0060] (1) All glass slides were pre-soaked in piranha solution overnight and then rinsed with ultrapure water.

[0061] (2) Silica microspheres with a diameter of 190 nm were centrifuged, and then repeatedly washed with anhydrous ethanol and centrifuged to form a silica ethanol suspension with a mass concentration of 1%.

[0062] (3) The glass slide soaked in piranha is placed vertically in the silica ethanol suspension. After 7 days, a silica colloidal crystal film is formed. The temperature of the environment is 25±5°C and the humidity is 10±2%.

[0063] The silica colloidal crystal film obtained in step S1 was scanned by a scanning electron microscope, and the results were as follows: Figure 1 As shown, Figure 1 This is a scanning electron microscope image of a silica colloidal crystal film. Figure 1 A is the front view of silica colloidal crystal film. Figure 1 B is a cross-sectional view of a silica colloidal crystal film. Figure 1 A shows that the silica microspheres are closely arranged in the front electron microscope photo, and there is no disorderly and irregular arrangement structure. Figure 1B shows that the silica microspheres are densely packed under the cross-sectional electron microscope photograph to form a typical colloidal crystal structure, indicating that the method described in step S1 successfully prepared a silica colloidal crystal film.

[0064] The reflection interference test of the silica colloidal crystal film obtained in step S1 is as follows: Figure 2 As shown in the figure, it can be seen that the silica colloidal crystal film has good interference fringes and obvious interference peaks, which is convenient for the subsequent calculation of optical thickness for EVs separation and detection.

[0065] S2. Modification of DSPE on silica colloidal crystal film

[0066] (1) The silica colloidal crystal film was immersed in a 0.1% APTES (aminopropyltriethoxysilane) ethanol solution (v / v) overnight for amination treatment.

[0067] (2) 1 mg of DSPE was dissolved in 1 mL of 30% ethanol solution (v / v) to obtain solution 1; 20 mg of NHS (N-hydroxysuccinimide) and 50 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 4 mL of Tris-HCl buffer (pH = 6.5) to obtain solution 2. Solution 1 and solution 2 were mixed and the pH value was adjusted to 7.5. The aminated silica colloidal crystal film was reacted with the mixed solution for 6 hours to obtain a DSPE-modified silica colloidal crystal film.

[0068] (3) The silica colloidal crystal film modified with DSPE obtained in step (2) is washed three times with ultrapure water and can be used for subsequent analysis or experiments. When not in use, it can be stored in ultrapure water for future use.

[0069] The DSPE-modified silica colloidal crystal film obtained in step S2 was subjected to reflection interference spectroscopy detection, and the results were as follows: Figure 3 As shown; Figure 3 It is the reflection interference spectrum of the same position on the silica colloidal crystal film before and after DSPE modification. It can be seen from the figure that after modification with DSPE, the reflection interference spectrum of the silica colloidal crystal film shifts to the right as a whole, proving that DSPE is successfully modified on the silica colloidal crystal film.

[0070] S3. Build OPLI system

[0071] (1) A hole was drilled in the silicone pad and covered on a glass slide with a silica colloidal crystal film modified with DSPE to form a reaction pool.

[0072] (2) Press the upper end with another glass slide to form a closed reaction pool.

[0073] (3) Fix the closed reaction pool obtained in step (2) on an inverted microscope and adjust the focus to the silica colloidal crystal layer.

[0074] (4) Through the spectroscope, couple the optical fiber and connect it to the spectrometer.

[0075] (5) Connect the catheter to the reaction pool and connect the catheter to the peristaltic pump so that the reaction liquid can flow into the hole in the middle of the silicone pad. Repeat the operation to connect another catheter so that the reacted liquid can flow out.

[0076] S4. Separation and enrichment of EVs and elution of EVs enriched in silica colloidal crystal films

[0077] A 0.1 M PBS solution is introduced into the closed reaction pool through the catheter in step S3 (5) at a flow rate of 0.4 mL / min by using a peristaltic pump, and a spectral line fitting is performed on the selected reflection interference spectrum band when the baseline of the spectrometer is stable. The spectral line fitting method is the extreme value method, and the optical thickness of the silica colloidal crystal film in real time is obtained by calculation; when the optical thickness of the silica colloidal crystal film is stable, 1 mL of MSC culture supernatant is circulated through the catheter in step S3 (5) by using a peristaltic pump, and a spectral line fitting is performed on the selected reflection interference spectrum band. The spectral line fitting method is the extreme value method, and the optical thickness of the silica colloidal crystal film is obtained by calculation (the optical optimum of the silica colloidal crystal film is within the range of 4000-6000 nm, but the initial thickness of the specific silica crystal film does not affect its thickness increment).

[0078] The DSPE molecules on the surface of the silica colloidal crystal film will specifically bind to the phospholipid bilayer of EVs, causing an increase in the optical thickness. After the curve rises steadily, the PBS solution is passed again to stabilize the baseline. Then, Triton X-100 or NP40 with a volume concentration of 0.02% is passed. As a detergent, it can wash away excess proteins and other impurities in the culture supernatant adsorbed by the silica colloidal crystal film. The PBS solution is passed again to stabilize the baseline. Finally, 1 mL of triethanolamine (TEA) solution is circulated to elute the EVs enriched in the silica colloidal crystal film and collect them in a centrifuge tube for subsequent experiments. The entire process of EVs separation is recorded by OPLI.

[0079] The results are as follows Figure 4As shown in the figure, after the baseline stabilized, the MSC culture supernatant was passed through the 200s cycle, and the optical thickness increased significantly. The curve rose rapidly during 200-1800s, and then stabilized. The separation was basically completed at 2000s. After washing with Triton X-100 or NP40, the optical thickness decreased slightly from 2200s to 2500s and stabilized quickly. After passing through the PBS solution, the optical thickness was still very stable, indicating that DSPE can stably bind to EVs. After passing through the TEA solution, the optical thickness decreased significantly and basically returned to the initial value, proving that the EVs adsorbed on the silica colloidal crystal film were completely eluted.

[0080] The optical thickness of the silica colloidal crystal film is calculated by using the extreme value method to fit the spectrum line, specifically according to the formula Fit the optical thickness, where λ is the real-time wavelength of the interference peak in the range of 430-1100 nm, k′ is the relative order, k′=0, 1, 2, 3, 4..., and nd is the optical thickness.

[0081] The EVs washed out after the optical thickness rise was completely stable were subjected to Western blotting experiments to analyze the expression of EVs-specific protein CD9. The results are as follows Figure 5 As shown in the gel image, CD9 is obviously expressed, indicating that the method (OPLI) described in the present invention can separate and enrich EVs in the supernatant of MSC culture fluid.

[0082] After the optical thickness rises completely and stabilizes, the eluted EVs are observed by transmission electron microscopy. Figure 6 As shown, many round structures can be clearly observed, and their sizes are consistent with the particle size range of EVs (30-150nm).

[0083] Example 2: Isolation and enrichment of EVs in urine

[0084] S1. Preparation of silica colloidal crystal film (SCC film)

[0085] (1) All glass slides were pre-soaked in piranha solution overnight and then rinsed with ultrapure water.

[0086] (2) Silica microspheres with a diameter of 190 nm were centrifuged, and then repeatedly washed with anhydrous ethanol and centrifuged to form a silica ethanol suspension with a mass concentration of 1%.

[0087] (3) The glass slide soaked in piranha is placed vertically in the silica ethanol suspension. After 7 days, a silica colloidal crystal film is formed. The temperature of the environment is 25±5°C and the humidity is 10±2%.

[0088] S2. Modification of DSPE on silica colloidal crystal film

[0089] (1) The silica colloidal crystal film was immersed in a 2% APTES (aminopropyltriethoxysilane) ethanol solution (v / v) overnight for amination treatment.

[0090] (2) 100 mg of DSPE was dissolved in 1 mL of 30% ethanol solution (v / v) to obtain solution 1; 200 mg of NHS (N-hydroxysuccinimide) and 400 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 4 mL of Tris-HCl buffer (pH = 6.5) to obtain solution 2. Solution 1 and solution 2 were mixed and the pH value was adjusted to 7.5. The aminated silica colloidal crystal film was reacted with the mixed solution for 6 hours to obtain a DSPE-modified silica colloidal crystal film.

[0091] (3) The silica colloidal crystal film modified with DSPE obtained in step (2) is washed three times with ultrapure water and can be used for subsequent analysis or experiments. When not in use, it can be stored in ultrapure water for future use.

[0092] S3. Build OPLI system

[0093] (1) A hole was drilled in the silicone pad and covered on a glass slide with a silica colloidal crystal film modified with DSPE to form a reaction pool.

[0094] (2) Press the upper end with another glass slide to form a closed reaction pool.

[0095] (3) Fix the closed reaction pool obtained in step (2) on an inverted microscope and adjust the focus to the silica colloidal crystal layer.

[0096] (4) Through the spectroscope, couple the optical fiber and connect it to the spectrometer.

[0097] (5) Connect the catheter to the reaction pool and connect the catheter to the peristaltic pump so that the reaction liquid can flow into the hole in the middle of the silicone pad. Repeat the operation to connect another catheter so that the reacted liquid can flow out.

[0098] S4. Use a peristaltic pump to pass 0.1 M PBS solution into the closed reaction pool through the catheter in step S3 (5) at a flow rate of 0.4 mL / min. After the baseline of the spectrometer is stable, perform spectral line fitting on the selected reflection interference spectrum band, and the spectral line fitting method is the extreme value method. After calculation, the optical thickness of the silica colloidal crystal film is obtained in real time; after the optical thickness of the silica colloidal crystal film is stable, use a peristaltic pump to circulate 1 mL of urine through the catheter in step S3 (5), and perform spectral line fitting on the selected reflection interference spectrum band. The spectral line fitting method is the extreme value method, and the optical thickness of the silica colloidal crystal film is obtained after calculation.

[0099] The DSPE molecules on the surface of the silica colloidal crystal film will specifically bind to the phospholipid bilayer of EVs, causing an increase in the optical thickness. After the curve rises steadily, the PBS solution is passed again to stabilize the baseline. Then, Triton X-100 or NP40 with a volume concentration of 0.02% is passed. As a detergent, it can wash away excess proteins and other impurities in the culture supernatant adsorbed by the silica colloidal crystal film. The PBS solution is passed again to stabilize the baseline. Finally, 1 mL of triethanolamine (TEA) solution is circulated to elute the EVs enriched in the silica colloidal crystal film and collect them in a centrifuge tube for subsequent experiments. The entire process of EVs separation is recorded by OPLI.

[0100] The results are as follows Figure 7 As shown in the figure, after the baseline stabilized, urine was introduced for 200 seconds, and the optical thickness increased significantly. The curve rose rapidly during 200-1800 seconds, and then stabilized. The separation was basically completed at 2000 seconds. After washing with Triton X-100 or NP40, the optical thickness decreased slightly from 2200 to 2500 seconds and stabilized quickly. After the PBS solution was introduced again, the optical thickness was still very stable, indicating that DSPE can stably bind to EVs. After the TEA solution was introduced, the optical thickness decreased significantly and basically returned to the initial value, proving that the EVs adsorbed on the silica colloidal crystal film were completely eluted.

[0101] The optical thickness of the silica colloidal crystal film is calculated by using the extreme value method to fit the spectrum line, specifically according to the formula Fitting optical thickness, where λ is the real-time wavelength of the interference peak in the range of 430-1100nm, that is, the selected reflection interference spectrum band, k′ is the relative order, k′=0, 1, 2, 3, 4..., and nd is the optical thickness.

[0102] The EVs washed out after the optical thickness rise was completely stable were subjected to Western blotting experiments to analyze the expression of EVs-specific protein CD9. The results are as follows Figure 8As shown in the gel image, CD9 is obviously expressed, indicating that the method (OPLI) described in the present invention can separate and enrich EVs in urine.

[0103] After the optical thickness rises completely and stabilizes, the eluted EVs are observed by transmission electron microscopy. Fig. 9 As shown in the figure, many round structures can be clearly observed, and their sizes are consistent with the particle size range of EVs.

[0104] Example 3: Validation of the effectiveness of real-time concentration detection of EVs

[0105] S1. Preparation of silica colloidal crystal film (SCC film)

[0106] (1) All glass slides were pre-soaked in piranha solution overnight and then rinsed with ultrapure water.

[0107] (2) Silica microspheres with a diameter of 190 nm were centrifuged, and then repeatedly washed with anhydrous ethanol and centrifuged to form a silica ethanol suspension with a mass concentration of 1%.

[0108] (3) The glass slide soaked in piranha is placed vertically in the silica ethanol suspension. After 7 days, a silica colloidal crystal film is formed. The temperature of the environment is 25±5°C and the humidity is 10±2%.

[0109] S2. Modification of DSPE on silica colloidal crystal film

[0110] (1) The silica colloidal crystal film was immersed in a 1% APTES (aminopropyltriethoxysilane) ethanol solution (v / v) overnight for amination treatment.

[0111] (2) 10 mg of DSPE was dissolved in 1 mL of 30% ethanol solution (v / v) to obtain solution 1; 50 mg of NHS (N-hydroxysuccinimide) and 100 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 4 mL of Tris-HCl buffer (pH = 6.5) to obtain solution 2. Solution 1 and solution 2 were mixed and the pH value was adjusted to 7.5. The aminated silica colloidal crystal film was reacted with the mixed solution for 6 hours to obtain a DSPE-modified silica colloidal crystal film.

[0112] (3) The silica colloidal crystal film modified with DSPE obtained in step (2) is washed three times with ultrapure water and can be used for subsequent analysis or experiments. When not in use, it can be stored in ultrapure water for future use.

[0113] S3. Build OPLI system

[0114] (1) A hole was drilled in the silicone pad and covered on a glass slide with a silica colloidal crystal film modified with DSPE to form a reaction pool.

[0115] (2) Press the upper end with another glass slide to form a closed reaction pool.

[0116] (3) Fix the closed reaction pool obtained in step (2) on an inverted microscope and adjust the focus to the silica colloidal crystal layer.

[0117] (4) Through the spectroscope, couple the optical fiber and connect it to the spectrometer.

[0118] (5) Connect the catheter to the reaction pool and connect the catheter to the peristaltic pump so that the reaction liquid can flow into the hole in the middle of the silicone pad. Repeat the operation to connect another catheter so that the reacted liquid can flow out.

[0119] S4. Use a peristaltic pump to pass 0.1M PBS solution into the closed reaction pool through the catheter in step S3 (5) at a flow rate of 0.4mL / min, wait for the baseline of the spectrometer to stabilize, perform spectral line fitting on the selected reflection interference spectrum band, the spectral line fitting method is the extreme value method, and the optical thickness of the real-time silica colloidal crystal film is obtained by calculation; wait for the optical thickness of the silica colloidal crystal film to stabilize, use a peristaltic pump to circulate 1mL of MSC culture supernatant through the catheter in step S3 (5), perform spectral line fitting on the selected reflection interference spectrum band, the spectral line fitting method is the extreme value method, and the optical thickness of the silica colloidal crystal film is obtained by calculation. The DSPE molecules on the surface of the silica colloidal crystal film will specifically bind to the phospholipid bilayer of EVs, causing the optical thickness to increase.

[0120] S5. Use PBS solution to dilute 1 mL of MSC culture supernatant in gradient concentrations (the dilution ratios include 0.1, 0.25, 0.5, 0.75 and 1), and repeat the process of step S4. Fig.10 As shown in the figure, the supernatant curve of the undiluted MSC culture medium (i.e., when the dilution ratio is 1) rises fastest, and the optical thickness change caused is also the highest. As the dilution ratio of the culture medium increases, the curve rises slower, and the optical thickness change caused also decreases.

[0121] The optical thickness of the silica colloidal crystal film is calculated by using the extreme value method to fit the spectrum line, according to the formula Fitting optical thickness, where λ is the real-time wavelength of the interference peak in the range of 430-1100nm, that is, the selected reflection interference spectrum band, k′ is the relative order, k′=0, 1, 2, 3, 4..., and nd is the optical thickness.

[0122] Fig.11The horizontal axis is the dilution ratio of the MSC culture supernatant, and the vertical axis is the change in optical thickness. Fig.11 It can be seen that by establishing a linear relationship between the dilution concentration of the culture supernatant and ΔOT, the linear relationship is very good, R 2 =0.991; indicating that the detection method provided by the present invention can effectively enrich EVs and realize real-time detection of concentration.

[0123] Example 4: Analysis of EVs membrane protein dynamics

[0124] S1. Preparation of silica colloidal crystal film (SCC film)

[0125] (1) All glass slides were pre-soaked in piranha solution overnight and then rinsed with ultrapure water.

[0126] (2) Silica microspheres with a diameter of 190 nm were centrifuged, and then repeatedly washed with anhydrous ethanol and centrifuged to form a silica ethanol suspension with a mass concentration of 1%.

[0127] (3) The glass slide soaked in piranha is placed vertically in the silica ethanol suspension. After 7 days, a silica colloidal crystal film is formed. The temperature of the environment is 25±5°C and the humidity is 10±2%.

[0128] S2. Modification of DSPE on silica colloidal crystal film

[0129] (1) The silica colloidal crystal film was immersed in a 1% APTES (aminopropyltriethoxysilane) ethanol solution (v / v) overnight for amination treatment.

[0130] (2) 10 mg of DSPE was dissolved in 1 mL of 30% ethanol solution (v / v) to obtain solution 1; 50 mg of NHS (N-hydroxysuccinimide) and 100 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 4 mL of Tris-HCl buffer (pH = 6.5) to obtain solution 2. Solution 1 and solution 2 were mixed and the pH value was adjusted to 7.5. The aminated silica colloidal crystal film was reacted with the mixed solution for 6 hours to obtain a DSPE-modified silica colloidal crystal film.

[0131] (3) The silica colloidal crystal film modified with DSPE obtained in step (2) is washed three times with ultrapure water and can be used for subsequent analysis or experiments. When not in use, it can be stored in ultrapure water for future use.

[0132] S3. Build OPLI system

[0133] (1) A hole was drilled in the silicone pad and covered on a glass slide with a silica colloidal crystal film modified with DSPE to form a reaction pool.

[0134] (2) Press the upper end with another glass slide to form a closed reaction pool.

[0135] (3) Fix the reaction cell on an inverted microscope and adjust the focus to the silica colloidal crystal layer.

[0136] (4) Through the spectroscope, couple the optical fiber and connect it to the spectrometer.

[0137] (5) Connect the catheter to the reaction pool and connect the catheter to the peristaltic pump so that the reaction liquid can flow into the hole in the middle of the silicone pad. Repeat the operation to connect another catheter so that the reacted liquid can flow out.

[0138] S4. Use a peristaltic pump to pass 0.1M PBS solution into the closed reaction pool through the catheter in step S3 (5) at a flow rate of 0.4mL / min, wait for the baseline of the spectrometer to stabilize, perform spectral line fitting on the selected reflection interference spectrum band, the spectral line fitting method is the extreme value method, and the optical thickness of the real-time silica colloidal crystal film is obtained by calculation; wait for the optical thickness of the silica colloidal crystal film to stabilize, use a peristaltic pump to circulate 1mL of MSC culture supernatant through the catheter in step S3 (5), perform spectral line fitting on the selected reflection interference spectrum band, the spectral line fitting method is the extreme value method, and the optical thickness of the silica colloidal crystal film is obtained by calculation. The DSPE molecules on the surface of the silica colloidal crystal film will specifically bind to the phospholipid bilayer of EVs, causing the optical thickness to increase.

[0139] S5. Pass 0.1M PBS solution, wait for the baseline to stabilize, then pass CD9 antibody solution. After the binding is stable, pass 0.1M PBS solution again. Fig.12 As shown. Fig.12 It can be seen that, taking the antibody binding of CD9 as an example, after EVs are fixed on the silica colloidal crystal film, the optical thickness gradually increases after the antibody solution is circulated. This is caused by the specific binding of the CD9 antibody to the EVs membrane protein CD9 fixed on the silica colloidal crystal film. When PBS is passed again, the optical thickness will slowly decrease, and there will be a slight dissociation process between CD9 and its antibody.

[0140] The optical thickness of the silica colloidal crystal film is calculated by using the extreme value method to fit the spectrum line, specifically according to the formula Fitting optical thickness, where λ is the real-time wavelength of the interference peak in the range of 430-1100nm, that is, the selected reflection interference spectrum band, k′ is the relative order, k′=0, 1, 2, 3, 4..., and nd is the optical thickness.

[0141] Repeat steps 1-6 with the antibody solutions of membrane proteins CD63 and CD81, and record the binding and dissociation curves of EVs membrane proteins and antibodies. The real-time optical thickness changes of EVs membrane proteins in the supernatant of MSC culture medium and the antibodies of membrane proteins CD9, CD63 and CD81 are shown in Figure 2. Fig.13 As shown. Fig.13 It can be seen that the optical thickness changes caused by the binding of membrane proteins CD9, CD63 and CD81 to their corresponding antibodies are not very different, and there is a slight dissociation phenomenon after passing into PBS, which proves that the binding between the two is very strong. The above results show that the method of the present invention can be used to detect the strength of binding between EV membrane proteins and antibodies, and can be used for drug screening.

[0142] Example 5: Liquid biopsy-assisted diagnosis of prostate cancer using EVs

[0143] S1. Preparation of silica colloidal crystal film (SCC film)

[0144] (1) All glass slides were pre-soaked in piranha solution overnight and then rinsed with ultrapure water.

[0145] (2) Silica microspheres with a diameter of 190 nm were centrifuged, and then repeatedly washed with anhydrous ethanol and centrifuged to form a silica ethanol suspension with a mass concentration of 1%.

[0146] (3) The glass slide soaked in piranha is placed vertically in the silica ethanol suspension. After 7 days, a silica colloidal crystal film is formed. The temperature of the environment is 25±5°C and the humidity is 10±2%.

[0147] S2. Modification of DSPE on silica colloidal crystal film

[0148] (1) The silica colloidal crystal film was immersed in a 1% APTES (aminopropyltriethoxysilane) ethanol solution (v / v) overnight for amination treatment.

[0149] (2) 10 mg of DSPE was dissolved in 1 mL of 30% ethanol solution (v / v) to obtain solution 1; 50 mg of NHS (N-hydroxysuccinimide) and 100 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 4 mL of Tris-HCl buffer (pH = 6.5) to obtain solution 2. Solution 1 and solution 2 were mixed and the pH value was adjusted to 7.5. The aminated silica colloidal crystal film was reacted with the mixed solution for 6 hours to obtain a DSPE-modified silica colloidal crystal film.

[0150] (3) The silica colloidal crystal film modified with DSPE obtained in step (2) is washed three times with ultrapure water and can be used for subsequent analysis or experiments. When not in use, it can be stored in ultrapure water for future use.

[0151] S3. Build OPLI system

[0152] (1) A hole was drilled in the silicone pad and covered on a glass slide with a silica colloidal crystal film modified with DSPE to form a reaction pool.

[0153] (2) Press the upper end with another glass slide to form a closed reaction pool.

[0154] (3) Fix the closed reaction pool obtained in step (2) on an inverted microscope and adjust the focus to the silica colloidal crystal layer.

[0155] (4) Through the spectroscope, couple the optical fiber and connect it to the spectrometer.

[0156] (5) Connect the catheter to the reaction pool and connect the catheter to the peristaltic pump so that the reaction liquid can flow into the hole in the middle of the silicone pad. Repeat the operation to connect another catheter so that the reacted liquid can flow out.

[0157] S4. Use a peristaltic pump to pass 0.1M PBS solution into the closed reaction pool through the catheter in step S3 (5) at a flow rate of 0.4mL / min, wait for the baseline of the spectrometer to be stable, perform spectral line fitting on the selected reflection interference spectrum band, the spectral line fitting method is the extreme value method, and the optical thickness of the real-time silica colloidal crystal film is obtained by calculation; wait for the optical thickness of the silica colloidal crystal film to be stable, use a peristaltic pump to circulate 1mL of prostate cancer patient urine through the catheter in step S3 (5), perform spectral line fitting on the selected reflection interference spectrum band, the spectral line fitting method is the extreme value method, and the optical thickness of the silica colloidal crystal film is obtained by calculation. The DSPE molecules on the surface of the silica colloidal crystal film will specifically bind to the phospholipid bilayer of EVs, causing the optical thickness to increase.

[0158] S5. Pass 0.1M PBS solution, wait for the baseline to stabilize, then pass CD9 antibody solution. After the binding is stable, use αvβ3 as a cancer marker and pass αvβ3 antibody solution, such as Fig.14 As shown. Fig.14 It can be seen that taking the αvβ3 cancer marker as an example, when the EVs in the urine of prostate cancer patients are fixed on the silica colloidal crystal film, the optical thickness gradually increases after the antibody solution is circulated. This is caused by the specific binding of the antibody to the αvβ3 membrane protein highly expressed in the EVs of prostate cancer patients fixed on the silica colloidal crystal film.

[0159] S6. Pass the antibody solution of αvβ3 and Trop-2 cancer markers, repeat steps 1-6, and record the changes in optical density of EVs binding to antibodies in urine samples of healthy subjects and prostate cancer patients.

[0160] The optical thickness of the silica colloidal crystal film is calculated by using the extreme value method to fit the spectrum line. Fitting optical thickness, where λ is the real-time wavelength of the interference peak in the range of 430-1100nm, that is, the selected reflection interference spectrum band, k′ is the relative order, k′=0, 1, 2, 3, 4..., and nd is the optical thickness.

[0161] The optical thickness changes caused by the αvβ3 and Trop-2 antibodies in the circulation and binding to the fixed EVs in the urine samples of prostate cancer patients are shown in Figure 2. Fig.14 and Fig.15 As shown. Fig.14 and Fig.15 It can be seen that the optical thickness of EVs in urine samples of prostate cancer patients increased rapidly during the process of fixation on silica colloidal crystal films, which were then washed with PBS. After the tumor marker antibodies αvβ3 and Trop-2 were circulated and pumped in, the optical thickness also increased significantly. Fig.16 It is the change in optical thickness caused by circulatory pumping of tumor marker antibody αvβ3 and Trop-2 solution after EVs were fixed in urine samples of prostate cancer patients and healthy people. The change in optical thickness caused by prostate cancer patients was significantly higher than that of healthy people, P<0.001, indicating that the method of the present invention can be used to assist liquid biopsy of prostate cancer.

[0162] Comparative Example 1: Isolation of EVs from MSC culture supernatant by ultracentrifugation

[0163] Ultracentrifugation (UC) method:

[0164] Take 1 mL of MSC culture medium. First, centrifuge at 2500g for 10 minutes and take the supernatant; then, centrifuge at 10000g at 4℃ for 45 minutes and take the supernatant; then, centrifuge at 120000g at 4℃ for 70 minutes and take the precipitate; finally, add PBS to the precipitate, disperse the precipitate, centrifuge at 120000g at 4℃ for 70 minutes and take the precipitate. Resuspend the precipitate with 1 mL of PBS.

[0165] The separation efficiency of the two methods was compared by analyzing the expression level of the EVs-specific protein CD9 obtained by the method of the present invention (OPLI) and the ultracentrifugation (UC) method. The results are as follows: Figure 5As shown in the bar graph, the capture intensity of EVs separated from the supernatant of MSC culture fluid by the method of the present invention is set to 1, and the capture intensity of EVs separated directly from the supernatant by ultracentrifugation is only 0.22, indicating that the efficiency of separating EVs from the supernatant of MSC culture fluid by the method of the present invention is significantly higher than the efficiency of separating EVs from the supernatant of MSC culture fluid by ultracentrifugation.

[0166] Comparative Example 2: Separation of EVs from urine by ultracentrifugation

[0167] Ultracentrifugation (UC) method:

[0168] Take 1 mL of MSC culture medium. First, centrifuge at 2500g for 10 minutes and take the supernatant; then, centrifuge at 10000g at 4℃ for 45 minutes and take the supernatant; then, centrifuge at 120000g at 4℃ for 70 minutes and take the precipitate; finally, add PBS to the precipitate, disperse the precipitate, centrifuge at 120000g at 4℃ for 70 minutes and take the precipitate. Resuspend the precipitate with 1 mL of PBS.

[0169] The separation efficiency of the two methods was verified by analyzing the expression of the EVs-specific protein CD9 obtained by the method of the present invention (OPLI) and the ultracentrifugation (UC) method. The results are as follows Figure 8 As shown in the bar graph, when the capture intensity of EVs separated from urine by the method of the present invention is set to 1, the capture intensity of EVs separated directly from urine by ultracentrifugation is only 0.22, indicating that the efficiency of separating EVs from urine by the method of the present invention is significantly higher than the efficiency of separating EVs from urine by ultracentrifugation.

Claims

1. A method for real-time enrichment and detection of extracellular vesicles, characterized in that: The method comprises the following steps: S1. Preparation of silica colloidal crystal film (SCC film) by vertical deposition method; S2. Preparation of DSPE-modified silica colloidal crystal films; S3. Set up OPLI (ordered porous layer interferometry) system: A reaction cell was assembled with DSPE-modified silica colloidal crystal film as the substrate, and an OPLI (ordered porous layer interferometry) system was formed with an inverted microscope and a fiber optic spectrometer. S4. Isolation and enrichment of extracellular vesicles: PBS solution is introduced into the reaction pool of the OPLI system. After the baseline is stabilized, the sample solution of the extracellular vesicles to be separated is recirculated. When the baseline displayed by the spectrometer is stable, the spectrum line is fitted for the selected reflection interference spectrum band. The optical thickness of the silica colloidal crystal film is calculated by extreme value fitting to obtain 1, which means that the separation and enrichment of the extracellular vesicles is completed. S5. Dissociation of enriched extracellular vesicles: After the enrichment is completed, detergent is introduced to wash away the impurities adsorbed on the SCC film; finally, the elution solution is circulated to dissociate the extracellular vesicles enriched in the SCC film; S6. Establish a linear relationship and analyze the concentration of extracellular vesicles: The sample solution of the extracellular vesicles to be separated at different dilution ratios is circulated, and steps S4 and S5 are repeated to establish a linear relationship with the optical thickness 1 as the ordinate and the dilution ratio as the abscissa to analyze the extracellular vesicle concentration; S7. Repeat steps S1-S5, except that in step S5, the elution solution is not passed, but the extracellular vesicle membrane protein antibody and PBS solution are passed; the selected reflection interference spectrum band is fitted with spectra, and the optical thickness 2 and optical thickness 3 of the silica colloidal crystal film are calculated by extreme value fitting method, and the expression level of the corresponding extracellular vesicle membrane protein and its binding and dissociation kinetics with the antibody are analyzed.

2. The method according to claim 1, characterized in that The method for preparing the DSPE-modified silica colloidal crystal film in step S2 comprises: The SCC film is immersed in an ethanol solution of aminopropyltriethoxysilane to obtain an amino-modified SCC film; DSPE is dissolved in an ethanol solution to obtain a solution 1; N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are dissolved in a Tris-HCl buffer to obtain a solution 2; the solution 1 and the solution 2 are mixed and the pH value is adjusted to obtain a mixed solution; the amino-modified SCC film is reacted with the mixed solution, and then washed with ultrapure water to obtain a DSPE-modified silica colloidal crystal film, which is stored in ultrapure water for later use.

3. The method according to claim 2, characterized in that The concentration of the aminopropyltriethoxysilane ethanol solution is 0.1%-2% (volume / volume), and the SCC film is immersed in the aminopropyltriethoxysilane ethanol solution for 1-48 hours.

4. The method according to claim 2, characterized in that: The dosage of the DSPE and ethanol solution is 1-100 mg: 1 mL.

5. The method according to claim 2, characterized in that: The dosage of the N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and Tris-HCl buffer is: 20-200 mg: 50-400 mg: 4 mL.

6. The method according to claim 2, characterized in that After the solution 1 and the solution 2 are mixed, the pH value is adjusted to 7.5, and the reaction time is 4-12 hours.

7. The method according to claim 1, characterized in that The OPLI system construction in step S3 includes the following steps: Drill a hole on the silicone pad and cover it on a glass slide of silica colloidal crystal film modified with DSPE to form a reaction pool; press the upper end with another glass slide to form a closed reaction pool; fix the reaction pool on an inverted microscope and adjust the focus to the silica colloidal crystal layer; couple the optical fiber through a spectroscope and connect it to a spectrometer; connect the catheter to the reaction pool and connect the catheter to a peristaltic pump so that the reaction liquid can flow into the hole in the middle of the silicone pad, repeat the operation to connect another catheter so that the reacted liquid can flow out.

8. The method according to claim 1, characterized in that The sample solution for separating extracellular vesicles in step S4 includes cell culture fluid, urine, emulsion, cerebrospinal fluid or blood.

9. The method according to claim 1, characterized in that: In step S5, the detergent comprises Triton or ethylphenyl polyethylene glycol, and the elution solution comprises triethanolamine.

10. The method according to claim 1, characterized in that The optical thickness in step S4 and step S7 is calculated according to the formula Fitting is performed, wherein λ is the real-time wavelength of the interference peak in the range of 430-1100 nm, that is, the selected reflection interference spectrum band, k′ is the relative order, k′=0, 1, 2, 3, 4..., and nd is the optical thickness.

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