Multi-channel paper chip device and application thereof in detection of small extracellular vesicle membrane protein

Abstract

The invention belongs to the technical field of biomedical engineering, and particularly relates to a multi-channel paper chip device and application of the multi-channel paper chip device in small cell outer vesicle membrane protein detection. A core component of the multichannel paper chip device disclosed by the invention is a paper chip jointly modified by a nucleic acid aptamer and a metal organic framework (MOF). According to the device, firstly, an MOF material grows on a paper chip in situ to provide amino modification sites of small extracellular vesicle membrane protein aptamers, meanwhile, non-specific adsorption of paper to impurity proteins is reduced, and the MOF material serves as a reference for ratio type detection. And then modifying an aptamer on the MOF so as to capture small extracellular vesicles expressing corresponding proteins through immune affinity. Meanwhile, a detection probe DP composed of the small extracellular vesicle membrane protein CD63 aptamer and the G-quadruplex is researched and developed, and the detection probe DP is used for colorimetric detection of the content of the captured small extracellular vesicles. The device can be used for detecting the small extracellular vesicle membrane protein through multiple channels, so that accurate diagnosis and typing of cancers are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical engineering, and in particular relates to a multi-channel paper chip device and an application thereof in the detection of small extracellular vesicle membrane proteins. Background Art

[0002] Liver cancer is one of the most common malignant tumors in the world, and its morbidity and mortality rates remain high all year round. In recent years, the understanding of liver cancer has become increasingly in-depth with the development of science and technology, but the mortality rate of liver cancer patients remains high. It is estimated that by 2025, more than 1 million people will be affected by liver cancer each year. The reason is that cancer is usually discovered in the middle and late stages, and the treatment effect is usually not ideal. At present, the focus and difficulty of liver cancer research is its early diagnosis. Among them, B-ultrasound examination and serum alpha-fetoprotein (AFP) determination are the main methods for liver cancer diagnosis. However, these detection methods often cannot detect the early symptoms of liver cancer in time, and can only detect the disease after morphological changes. Therefore, it is urgent to explore and develop new biomarkers to provide new ideas for improving the diagnosis and treatment of liver cancer.

[0003] Biomarkers refer to indicators that can objectively measure and evaluate the physiological or pathological processes of organs, tissues, cells, and even organelles. They are widely present in human body fluids such as blood and tissue fluid, and abnormalities in their expression levels are usually closely related to the occurrence and development of cancer. Among them, small extracellular vesicles (sEVs) have become the most promising biomarkers for early diagnosis and treatment monitoring of cancer due to their high abundance, ubiquity, and excellent stability in various body fluids. Therefore, the development of excellent biomarker detection technology based on small extracellular vesicles is of great significance for the diagnosis and treatment of cancer, reducing the mortality rate of its patients, and conducting basic research related to cancer.

[0004] Small extracellular vesicles are a new biomarker for cancer diagnosis, which can deeply reveal the phenotypic information of organisms. Therefore, the immediate detection of small extracellular vesicles is of great significance for large-scale screening of cancer. However, the current methods for the separation of small extracellular vesicles and the analysis of small extracellular vesicle surface membrane proteins are still limited by cumbersome operations and expensive large instruments, making it difficult to promote their use. Based on this, there is an urgent need to develop a low-cost, cost-effective and easy-to-operate small extracellular vesicle detection method. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the present invention proposes a multi-channel paper chip device and a small extracellular vesicle membrane protein detection method, which can measure the protein expression profiles of small extracellular vesicle membranes in culture fluids of various cancer cells and serum of cancer patients at a relatively low cost, and realize accurate diagnosis and typing of cancer.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides a multi-channel paper chip device (Exopp-PAD), the core components of which are a variety of paper chips C@MOF@Paper modified by aptamers, wherein the C@MOF@Paper includes MOF@Paper on which UiO-66-NH2 is in situ grown on filter paper and nucleic acid aptamers, and the nucleic acid aptamers are used to specifically recognize small extracellular vesicle membrane proteins.

[0008] Preferably, the preparation method of the device is: opening a plurality of holes on an endurance board, filling different C@MOF@Paper into each hole as a test point, and filling MOF@Paper without aptamer modification as a reference point, then coating the endurance board with a filter membrane, and then loading it into a replaceable membrane filter, and then connecting both ends of the filter to a syringe to prepare a multi-channel paper chip device.

[0009] Preferably, the nucleic acid aptamer includes the EpCAM aptamer shown in SEQ ID No: 2, the PTK7 aptamer shown in SEQ ID No: 3, the CEA aptamer shown in SEQ ID No: 4, and the PD-L1 aptamer shown in SEQ ID No: 5.

[0010] Preferably, the preparation method of MOF@Paper comprises the following steps:

[0011] S1. Dissolve ZrCl4 in a mixture of formic acid and ethanol, then place filter paper in the resulting mixture and let it stand to allow the surface of the paper to fully absorb Zr 4+ ion,

[0012] S2. After dissolving BDC-NH2 in a mixture of formic acid, ethanol and water, the BDC-NH2 solution is added to the solution of step S1, and the solution is allowed to stand at room temperature on the surface of the paper to generate MOF nanoparticles, which are then washed and dried to obtain MOF@Paper.

[0013] More preferably, the filter paper is Whatman No. 4 filter paper.

[0014] More preferably, in the mixture of formic acid and ethanol, the volume ratio of formic acid to ethanol is 6-9:20; in the mixture of formic acid, ethanol and water, the volume ratio of formic acid, ethanol and water is 6-9:20:7-10.

[0015] The second aspect of the present invention provides a kit for detecting small extracellular vesicle membrane proteins, the kit comprising the multi-channel paper chip device described in the first aspect and a detection probe DP, wherein the detection probe DP has a nucleotide sequence as shown in SEQ ID No: 1.

[0016] The present invention in situ grows a MOF material with amino groups on filter paper, and further modifies a series of aptamers that can specifically identify small extracellular vesicle membrane proteins on the MOF surface to obtain a C@MOF@Paper paper chip. While the paper chip has the ability to specifically capture small extracellular vesicles, it can also improve the filter paper's anti-protein nonspecific adsorption performance through the special surface properties of the MOF material, and the color of the MOF itself can be used as a reference for subsequent ratio detection. At the same time, a DP nucleic acid probe is prepared using a G-quadruplex sequence and a CD63 aptamer sequence. Based on the affinity reaction of the aptamer and the specific binding with the small extracellular vesicle membrane protein CD63, the structure of DP is changed, thereby releasing the catalytic color development activity of the G-quadruplex. Then it is combined with Hemin and exhibits peroxidase catalytic activity to catalyze TMB color development, obtaining a colorimetric detection signal of small extracellular vesicles, thereby realizing specific recognition and detection of small extracellular vesicle membrane proteins. In addition, by integrating paper chips targeting multiple membrane proteins into a multi-channel chip and performing capture, washing and detection operations through syringes, the expression profile of small extracellular vesicle membrane proteins can be measured simply and cheaply.

[0017] Preferably, the method of using the kit is as follows: use the syringe at one end of the multi-channel paper chip device to absorb the body fluid sample to be tested, slowly push it, and pull the syringe at the other end at the same time to make the liquid completely flow into the syringe at the other end, and repeat this cycle to allow the sample to be fully combined with the device, and then replace the body fluid sample to be tested with washing solution, DP detection solution, and Hemin solution in turn, and operate in the same way, and finally take out the multi-channel paper chip device, add TMB single-component colorimetric solution to each test point and reference point, observe the color development results, take pictures, and then analyze the Hue value for quantitative analysis.

[0018] More preferably, the body fluid sample to be tested is a serum sample.

[0019] More preferably, the quantitative analysis method is: utilizing the self-calibration property that the color of the reference point remains unchanged, constructing a ratiometric Hue value detection signal: R-ΔHue=ΔHue sample / ΔHue reference, ΔHue=Hue after reaction-Blank Hue, and then using R-ΔHue and the logarithm of the small extracellular vesicle concentration as a standard curve.

[0020] More preferably, the solid board is a PC solid board.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention discloses a multi-channel small extracellular vesicle membrane protein detection method and a matching paper chip device (Exopp-PAD), which realizes cancer cell typing by detecting the expression spectrum of small extracellular vesicle membrane proteins in body fluid samples. The core component of the device is a paper chip modified with an aptamer and a metal organic framework (MOF). The preparation method of the device is to first grow MOF material in situ on a paper chip to provide amino modification sites for small extracellular vesicle membrane protein aptamers, while reducing the nonspecific adsorption of paper to impurity proteins, and serving as a reference for ratiometric detection. The aptamer is then modified on the MOF to capture small extracellular vesicles expressing the corresponding protein by immune affinity. At the same time, a detection probe (DP) composed of a small extracellular vesicle membrane protein CD63 aptamer and a G-quadruplex was developed for colorimetric detection of the captured small extracellular vesicle content. In addition, paper chips targeting different membrane protein targets were assembled into a multi-channel capture chip, and the syringe controlled the interaction between the sample, washing solution, DP detection solution and the paper chip to complete the capture, washing and detection of small extracellular vesicles. It can be seen that using this device, the protein expression profile of small extracellular vesicle membranes in a variety of cancer cell culture fluids and cancer patient serum can be determined at a low cost, and the device is expected to achieve accurate diagnosis and typing of cancer.

[0023] Specifically, the present invention has the following advantages: (1) The aptamer-based paper chip and detection probe are low-cost and stable in performance, which can improve the cost-effectiveness and stability of detection and diagnosis; (2) Metal-organic framework modification can improve the capture efficiency and purity of the paper chip and improve the detection performance; (3) The developed paper chip device does not require expensive and complex equipment and professional skills, and can detect small extracellular vesicle membrane proteins in multiple channels, thereby achieving accurate typing diagnosis of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 (a) Physical picture of the small extracellular vesicle separation and analysis device; (b) Schematic diagram of paper chip preparation; (c) Schematic diagram of DP detection; (d) Schematic diagram of Exopp-PAD for small extracellular vesicle capture and membrane protein detection;

[0025] Figure 2 SEM images of (a, b) unmodified paper chip (Paper) and (c, d) MOF-modified paper chip (MOF@Paper);

[0026] Figure 3 (a) PXRD spectra of MOF@Paper, Paper, MOF powder and standard UiO-66-NH2; (b) ATR-FTIR spectra of MOF@Paper, Paper and MOF powder;

[0027] Figure 4(a) SEM image of MOF@Paper and (b) EDS element distribution map of MOF@Paper;

[0028] Figure 5 (a) Photos of unmodified paper chip and MOF@Paper before and after BSA adsorption and CBB reaction; (b) Hue value and (c) ΔHue value obtained from each sample in (a);

[0029] Figure 6 (a) PXRD spectra of aptamer-modified MOF@Paoper (C0@MOF@Paper) and MOF@Paper without aptamer modification; (b) ATR-FTIR spectra of C0@MOF@Paper and MOF@Paper;

[0030] Figure 7 (a) Photos of MOF@Paper without EDC / NHS activation and MOF@Paper activated with EDC / NHS before and after color development reaction with TMB after fixing the G quadruplex sequence; (b) A bar graph of the Hue values ​​obtained from each sample in (a);

[0031] Figure 8 Capture SEM images of small extracellular vesicles for C1@MOF@Paper;

[0032] Fig. 9 Circular dichroism spectra of DP reacting with CD63 protein, small extracellular vesicles and blank control;

[0033] Fig.10 is the fluorescence spectrum of DP reacting with CD63 protein, small extracellular vesicles and blank control;

[0034] Fig.11 The UV-visible absorption spectra of DP reacting with CD63 protein, small extracellular vesicles and blank control;

[0035] Fig.12 (a) Optimization of Hemin concentration; (b) Optimization of incubation time of Hemin and G4;

[0036] Fig.13 is the relationship between the concentration of small extracellular vesicles and absorbance;

[0037] Fig.14 (a) The color development results of different concentrations of small extracellular vesicles combined with DP on C@MOF@Paper; (b) The relationship between ΔHue value and small extracellular vesicle concentration;

[0038] Fig.15(a) Schematic diagram of Exopp-PAD assembly; (b) Color development results of different types of small extracellular vesicles after binding to DP in Exopp-PAD;

[0039] Fig.16 (a) The color development results of different concentrations of small extracellular vesicles combined with DP in Exopp-PAD and the relationship between R-ΔHue and (b) the logarithm of different concentrations of HepG2 (i), HuH-7 (ii), and LO2 (iii);

[0040] Fig.17 is the R-ΔHue value of each target protein on HepG2 / HuH-7 / LO2 small extracellular vesicles;

[0041] Fig.18 (a) Photograph of actual sample after analysis using Exopp-PAD; (b) Heat map of protein expression in actual sample. DETAILED DESCRIPTION

[0042] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0043] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0044] The present invention provides a multi-channel paper chip device and its application in the detection of small extracellular vesicle membrane proteins, including the following contents:

[0045] 1. Construction of a small extracellular vesicle separation, enrichment and analysis device

[0046] Amino-modified MOF materials were grown in situ on filter paper, and a series of aptamers that can specifically recognize small extracellular vesicle membrane proteins were further modified on the MOF surface to obtain C@MOF@Paper (c represents nucleic acid aptamer), which has the function of specifically capturing small extracellular vesicles. The obtained series of composite paper chips were then assembled on an acrylic plate to form Exopp-PAD, which was then assembled with two polycarbonate (PC) filter membranes with a pore size of 200nm in a replaceable membrane filter, and then the filter was connected to a syringe to construct a small extracellular vesicle separation, enrichment and analysis device, such as Figure 1 As shown in a and b.

[0047] 2. Design of detection probe DP and validation analysis of its binding to small extracellular vesicles

[0048] (1) Figure 1 As shown in Figure c, a hairpin detection probe DP was designed. The probe was composed of a G-quadruplex sequence connected to a CD63 aptamer sequence. The sequence was used to specifically bind to the CD63 protein highly expressed on the surface of small extracellular vesicles. After binding, the probe structure changed and released the G-quadruplex sequence. The content of each protein in the small extracellular vesicles captured by Exopp-PAD could be reflected by detecting the content of G-quadruplex.

[0049] (2) The fluorescence emitted by G-quadruplex after binding to ThT under excitation at a specific wavelength and the peroxidase catalytic activity exhibited after forming a complex with Hemin were used to verify the presence of G-quadruplex through circular dichroism spectroscopy, fluorescence spectroscopy and UV-visible spectroscopy, and to examine the binding of DP to CD63 and small extracellular vesicles.

[0050] (3) Taking advantage of the fact that the G-quadruplex sequence exhibits peroxidase catalytic activity after binding to Hemin, different concentration gradients of Hemin and different reaction temperature gradients were set to optimize the binding conditions of the G-quadruplex sequence and Hemin.

[0051] (4) Linear analysis of the color development results after the detection probe DP binds to the SVs was performed on a 96-well plate and C@MOF@Paper, respectively. Different concentrations of SV suspensions were added, and then reacted with the detection probe DP, Hemin, and TMB in sequence. For the 96-well plate, the absorbance was detected at 595 nm using an ELISA reader, and a standard curve was drawn. For C@MOF@Paper, the Hue value was recorded and read by taking a photo, and a standard curve of the Hue value and the SV concentration was drawn.

[0052] 3. Multi-channel detection of small extracellular vesicle proteins based on Exopp-PAD

[0053] Exopp-PAD and detection probe DP were used to test the color development results of HepG2, HuH-7 and LO2 small extracellular vesicles with known concentrations, and photos were taken and the Hue values ​​were analyzed using ImageJ for quantitative analysis. The specific process diagram is shown in the figure. Figure 1 As shown in d.

[0054] In order to further clearly present the process of constructing a multi-channel paper chip device and detecting small extracellular vesicle membrane proteins of the present invention, a detailed description is given below in conjunction with examples.

[0055] 1. Preparation and characterization of MOF@Paper

[0056] (1) In-situ growth of MOF on paper chips

[0057] Cut Whatman No. 4 filter paper into 6 mm diameter circular pieces by punching. Dissolve 0.5 mmol ZrCl4 in a mixture of 0.7 mL formic acid and 2.0 mL ethanol. After ultrasonication for 1 h, add the paper chip to the ZrCl4 solution and let it stand for 2 h to allow the surface of the paper chip to fully adsorb Zr 4+ ions. On the other hand, 0.2mmol BDC-NH2 was dissolved in a mixture of 0.7mL formic acid, 2.0mL ethanol and 0.8mL pure water, and ultrasonically treated for 2h. After the ultrasonic treatment, the BDC-NH2 solution was added to the ZrCl4 solution containing the paper chip, and after slight shaking and mixing, it was allowed to react at room temperature for 8h to generate MOF nanoparticles on the surface of the paper chip. After that, it was washed with anhydrous ethanol and water for 3 times respectively, and dried at 37°C for 10min to obtain the UiO-66-NH2 functionalized paper chip (MOF@Paper), which was finally placed in a sealed bag for storage.

[0058] (2) SEM and spectroscopy characterization of paper chips

[0059] The filter paper before MOF modification (Paper) and the filter paper after MOF modification (MOF@Paper) were adhered to the sample stage with conductive glue, vacuum dried in a vacuum drying oven for 12 h, and gold was sprayed on the sample with a particle sputtering instrument for 60 s, and then characterized by SEM at an accelerating voltage of 3 kV. Figure 2 As shown, SEM results show that the paper chip shows an interlaced porous fiber structure before and after the growth of MOF, such as Figure 2 a and 2c, that is, the growth of MOF does not change the porous three-dimensional structure of paper itself. Compared with the smooth fibers of paper itself ( Figure 2 b), MOF@paper forms uniform nanoparticles on the fiber surface ( Figure 2 d). SEM characterization shows that after modification with UiO-66-NH2, MOF@Paper still retains the original porous structure and filtration function of paper, allowing the sample liquid to pass through the paper chip, ensuring the ability to fully contact and interact with the aptamer subsequently modified on the paper chip.

[0060] (3) PXRD and ATR-FTIR characterization of paper chips

[0061] In order to verify that the nanoparticles generated on the paper surface are the target MOF material UiO-66-NH2, X-ray powder diffraction characterization was performed on Paper, MOF@Paper and pure MOF powder respectively. The experimental parameters were: scanning speed 6° per minute, scanning range 5° to 40°, step length 0.02°. The results are shown in Figure 2. Figure 3As shown in a, MOF@Paper exhibits characteristic PXRD peaks of Paper and UiO-66-NH2 (111) and (002) located at 2θ = 7.4° and 8.5°, indicating that the structure generated in the synthesized MOF@Paper is UiO-66-NH2.

[0062] At the same time, ATR-FTIR was used to perform infrared tests on Paper, MOF@Paper and synthesized MOF powder. The experimental parameters were: scanning range 500cm -1 Up to 2500cm -1 , step length 1cm -1 , scan 16 times. Figure 3 As shown in b, MOF@Paper exhibits the characteristic absorption peak of UiO-66-NH2: 768 cm -1 The peak at 1374 cm is attributed to Zr-O stretching vibration. -1 and 1567cm -1 The peak at is attributed to the stretching vibration of the carbonyl group. The above results further illustrate that UiO-66-NH2 nanoparticles were successfully grown in situ on paper fibers.

[0063] In addition, if Figure 4 As shown, the SEM image of MOF@Paper ( Figure 4 a) and EDS element distribution diagram ( Figure 4 b) shows the extensive distribution of Zr on its surface, indicating that UiO-66-NH2 nanoparticles grow uniformly and extensively on paper fibers. The above results confirm that UiO-66-NH2 nanoparticles grow and are widely and uniformly distributed on paper fibers, providing amino active sites that are easily modified for paper sheets.

[0064] (4) Study on the ability of paper chips to resist nonspecific protein adsorption

[0065] Using BSA as a protein model molecule, Paper and MOF@Paper (6 mm diameter disc) were first incubated with BSA solution (20 μL, 10 mg / mL) (room temperature for 20 minutes) and washed thoroughly. Then, Coomassie Brilliant Blue solution (10 μL, 1 μM) was added and reacted at room temperature for 30 minutes to develop the BSA adsorbed on the paper. The results are shown in Figure 2. Figure 5 As shown in a. Paper changed color significantly, while MOF@Paper hardly changed color. Further quantification of the color change Hue value using ImageJ software revealed that the ΔHue value of Paper was 10.6 times that of MOF@Paper ( Figure 5 b, Figure 5c). The above results indicate that a large amount of BSA is nonspecifically adsorbed on the surface of unmodified paper fibers, while UiO-66-NH2 modification can greatly reduce the nonspecific adsorption of protein by paper.

[0066] 2. Aptamer modification on paper chip and its characterization

[0067] EDC-NHS was used to couple the amino groups on the paper chip with the aptamer modified with the carboxyl group at the 5' end. Specifically, 1.0mmol NHS and 4.0mmol EDC were dissolved in 10mL of water to prepare a mixed solution of 0.1M NHS and 0.4M EDC, and then MOF@Paper was immersed in the newly prepared EDC-NHS solution for 10min, and then washed with anhydrous ethanol and water for 3 times, and then immersed in 4 5mL1.0μM capture aptamers (C1-C4) and reference (Ctrl) solutions for 3h in batches, and then washed with PBST for 3 times, and dried at 37℃ for 15min to obtain the aptamer-modified paper chip (C@MOF@Paper, C1-C4 are nucleic acid aptamers of EpCAM, PTK7, CEA and PD-L1, respectively, as shown in Table 1). Finally, it was placed in a sealed bag and stored at -20℃ for future use. MOF@Paper and C@MOF@Paper (C1@MOF@Paper was used as the paper chip for feasibility verification) were characterized by PXRD and ATR-FTIR.

[0068] In order to verify that the carboxyl-modified aptamer can be successfully fixed on MOF@Paper, the 5'-terminal carboxyl-modified G-quadruplex sequence C0 with mimetic enzyme activity and covalent bonding function was combined with MOF@Paper through EDC / NHS to obtain C0@MOF@Paper for relevant characterization. First, PXRD and ATR-FTIR characterization were performed on C0@MOF@Paper. The results are as follows: Figure 6 Compared with the MOF@Paper without incubation of aptamer, the PXRD patterns of the two are basically consistent ( Figure 6 a), proving that the structure of MOF did not change after modification of the aptamer. Figure 6 b) It can be seen that C0@MOF@Paper has a peak at 1640 cm -1 There is a new infrared absorption peak at , which should be attributed to the characteristic peak of the carbonyl group in the amide bond. This is because the carboxyl group on C0 undergoes an amidation reaction with EDC / NHS to form an amide bond. The above results can preliminarily prove that the aptamer can be successfully fixed on MOF@Paper under the bridging of EDC / NHS.

[0069] After forming a complex with Hemin, the G-quadruplex sequence can show peroxidase catalytic activity, and then catalyze TMB / H2O2 for color development reaction. Based on this, a probe G4 (1.0μM, 5mL) directly composed of a G-quadruplex sequence and modified with a 5'-end carboxyl group was designed, and then it was incubated with MOF@Paper with and without EDC / NHS activation at room temperature for 3h. After washing, Hemin solution (4μM, 20μL) was added for incubation (37℃, 30 minutes), and after sufficient washing, TMB color development solution (1mM, 20μL) was added for reaction (37℃, 30 minutes), and the color change was observed and photographed ( Figure 7 a). Then use ImageJ software to quantify its Hue value ( Figure 7 b), it can be found that after EDC / NHS activation treatment, the paper chip finally changes color significantly, while the paper chip without EDC / NHS activation treatment basically does not undergo color development. The above results further indicate that nucleic acid sequences can be covalently modified on MOF@Paper through EDC / NHS reaction, and the G-quadruplex sequence modified on MOF@Paper can catalyze color development on paper.

[0070] Table 1 Nucleotide sequences used in the experiment

[0071]

[0072] 3. Validation of paper chip loading and enrichment of small extracellular vesicles

[0073] Exosomes were collected from HepG2 cell culture medium by ultracentrifugation. After the cells grew to about 70% of the area of ​​the cell culture flask, the culture medium was replaced with DMEM medium containing 1.5% exosome-free FBS and cultured for 48 h. Afterwards, the cell culture suspension was collected and transferred to a centrifuge tube, avoiding the cell pellet, and centrifuged at 300g for 10 min at 4°C to remove large particles and cells. The supernatant was then transferred to a new centrifuge tube and centrifuged at 2000g for 20 min at 4°C to remove cell debris and larger particles. Subsequently, the supernatant was transferred to a new centrifuge tube and centrifuged at 10000g for 30 min at 4°C to remove large vesicles and protein aggregates. The obtained supernatant was centrifuged at 4°C and 120,000g for 90 min, and the supernatant was carefully removed with a pipette. The exosome pellet was washed with PBS and resuspended, and then centrifuged again at 4°C and 120,000g for 90 min. The supernatant was then carefully removed, and 200 μL PBS was added to resuspend the exosome pellet, and 10 μL of each was aliquoted and stored at -80°C for later use.

[0074] Take 10 μL of HepG2 small extracellular vesicles and drop them on C@MOF@Paper (take C1 modified paper chip as the paper chip for feasibility verification), incubate at room temperature for 1 hour, wash off the unbound small extracellular vesicles with PBST, and stick the paper chip on the SEM sample stage with conductive glue after drying, vacuum dry for 12 hours, and characterize it with SEM. SEM is used to verify whether C@MOF@Paper captures small extracellular vesicles. Taking the SEM characterization in C1@MOF@Paper as an example, the results are as follows Figure 8 As shown in the figure, it can be seen that after combining with small extracellular vesicles, the MOF on the paper fiber captured round nanovesicles smaller than 200 nm. This result shows that the device can load and enrich small extracellular vesicles.

[0075] 4. Feasibility verification of the binding of the detection probe to CD63 and small extracellular vesicles

[0076] (1) Circular dichroism spectroscopy verification

[0077] DP solution (1 μM, 20 μL) was mixed with 20 μL of recombinant CD63 protein (1 μg / mL), small extracellular vesicles (Exos, 10 8 particles / mL) and PBS buffer solution (0.1 mM) were incubated (37°C, 30 minutes) and circular dichroism spectrometry was performed. The results are shown in Fig. 9 Compared with the blank control, when CD63 and small extracellular vesicles are present, the circular dichroism spectrum of the detection probe DP has a negative peak at 245nm and a positive peak at 270nm. This is because a parallel G-quadruplex spatial structure is formed, proving the feasibility of DP binding to CD63 and small extracellular vesicles.

[0078] (2) Fluorescence spectroscopy verification

[0079] An equal amount of thioflavin T (ThT, 10 μM, 20 μL) solution was added to the solution after DP was incubated with recombinant CD63 protein, small extracellular vesicles and blank control, incubated (37°C, 30 min) and diluted to 100 μL with PBS, and then the fluorescence emission spectrum was measured under 425 nm excitation light. Fig.10 Compared with the blank control, after DP was combined with CD63 protein and small extracellular vesicles, there was a fluorescence emission peak at 495nm in the presence of ThT, which further confirmed the feasibility of DP combining with CD63 and small extracellular vesicles.

[0080] (3) UV-Vis absorption spectrum verification

[0081] After DP was incubated with recombinant CD63 protein, small extracellular vesicles and blank control, an equal amount of Hemin (Hemin solution) solution was added to the solution, incubated (37°C, 30 minutes) and TMB single-component colorimetric solution was added to fully react, and then HCl solution was added to terminate the color development, and the UV-visible absorption spectrum was measured. Fig.11 As shown. Compared with the blank control, after DP was combined with CD63 protein and small extracellular vesicles, incubated with Hemin and added with TMB for color development, there was a strong absorption peak at 450nm. This also further verified the feasibility of DP combining with CD63 and small extracellular vesicles.

[0082] 5. Optimization of experimental conditions for binding of G-quadruplex to Hemin

[0083] G-quadruplex nucleic acid (G4, see Table 1) was dissolved in 1×TE Buffer and diluted to 10.0 μM. Hemin powder was dissolved in PBS buffer and diluted to 50.0 μM for later use. First, 10 mixed solutions with a volume of 80 μL, a G4 concentration of 1.0 μM, and a Hemin concentration of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 μM were prepared in a 96-well plate. After incubation at room temperature for 30 minutes, 20 μL of TMB single-component colorimetric solution was added. After reacting at room temperature for 20 minutes, 20 μL of 1.0 M HCl was added to terminate the reaction. The absorbance at 450 nm was then measured with an enzyme reader. The results are as follows: Fig.12 As shown in a. The absorbance first increases with the increase of Hemin concentration. When the Hemin concentration exceeds 4.0 μM, the absorbance has no obvious change, so the optimal concentration of Hemin is 4.0 μM.

[0084] Using the optimized Hemin concentration in the previous step, the incubation time of G4 and Hemin was set to 10, 20, 30, 60, and 90 min, respectively. After the incubation, 20 μL of TMB single-component colorimetric solution was added. After reacting at room temperature for 20 min, 20 μL of 1.0 M HCl was added to terminate the reaction. The absorbance at 450 nm was then measured with an ELISA reader and plotted, as shown in Figure 2. Fig.12 As shown in b, the absorbance increased with the incubation time, and there was no significant change in the absorbance after 30 min, so the optimal incubation time of Hemin and G-quadruplex was 30 min.

[0085] 6. Linear analysis of the color development results after the detection probe DP binds to small extracellular vesicles

[0086] HepG2 small extracellular vesicles were diluted with PBS to concentrations of 0, 1, 5, 10, 50, 100×10 6 pc·mL -120 μL of each dilution was added to a 96-well plate, and then mixed with 20 μL of 1.0 μM DP solution for 45 min, and then 20 μL of 4.0 μM Hemin solution was added for 30 min, washed 3 times with PBST, and then 20 μL of TMB single-component colorimetric agent was added for 20 min. The absorbance was then detected at 595 nm using an ELISA instrument, and a standard curve was obtained based on the concentration and absorbance of small extracellular vesicles: Abs. = 0.0673 × lg [Exos] – 0.0383 (R 2 =0.98), such as Fig.13 As shown. It can be seen that when the DP concentration remains unchanged and other conditions remain the same, the absorbance of the solution is well linearly correlated with the logarithm of the small extracellular vesicle concentration.

[0087] Subsequently, the linear relationship between DP and small extracellular vesicles on C@MOF@Paper was tested. 6 pc·mL -1 ) were added to C@MOF@Paper (6 mm diameter disc) with 20 μL of HepG2 small extracellular vesicle dilutions. After incubation for 1 hour, the disc was washed with PBST three times, and 20 μL of 1.0 μM DP solution was added for 45 minutes. Then 20 μL of 4.0 μM Hemin solution was added for 30 minutes. The disc was washed with PBST three times, and then 20 μL of TMB single-component color developer was added for 20 minutes. The color change after the reaction was recorded by taking a photo (14a), and a standard curve was obtained based on the Hue value and the small extracellular vesicle concentration: ΔHue = 8.03 × lg [Exos] – 7.11 (R 2 =0.96), such as Fig.14 As shown in b. It can be seen that when the DP concentration remains unchanged and other conditions remain the same, the ΔHue value of the paper chip color change is well linearly correlated with the logarithm of the small extracellular vesicle concentration.

[0088] 7. Multi-channel detection of small extracellular vesicle proteins based on Exopp-PAD

[0089] First, a device for enriching small extracellular vesicles and analyzing small extracellular vesicle proteins was constructed. The device includes three main components: a syringe, a replaceable membrane filter with a diameter of 25 mm, and a vertical small extracellular vesicle enrichment and membrane protein detection chip (Exopp-PAD). The specific preparation method is as follows: 5 holes with a diameter of 6 mm were dug out on a PC endurance board with a diameter of 25 mm and a thickness of 0.2 mm, and the paper chip C@MOF@Paper modified with 4 pre-prepared aptamers (Table 1) was filled as 4 test points, and MOF@Paper without aptamer modification was filled as a reference point to form Exopp-PAD, which was then coated with two filter membranes with a diameter of 25 mm and a pore size of 200 nm, and then loaded into a replaceable membrane filter with a diameter of 25 mm, and then the two ends of the filter were connected to the syringe. When working, use one end of the syringe to absorb 2mL of sample liquid, push slowly, and pull the other end at the same time. When the liquid completely flows into the other end, repeat this operation, and repeat this cycle to make Exopp-PAD fully combine with the sample. Then replace the sample liquid with washing liquid, DP detection liquid, and Hemin solution in the same way.

[0090] Exopp-PAD and DP were used to test the color development results of HepG2, HuH-7 and LO2 small extracellular vesicles with known concentrations, respectively, to establish standard curves of EpCAM, PTK7, CEA and PD-L1 on small extracellular vesicles of each type of cells. The specific process is as follows: different concentrations of liver / hepatoma cell-derived small extracellular vesicles (0, 5.0×10 6 –5.0×10 8 pc·mL -1 ) was added to 50% FBS in PBS to prepare the serum sample of small extracellular vesicles. Fig.15 a, and then the small extracellular vesicle serum sample is introduced into the Exopp-PAD through a syringe. When the sample passes through the filter, the membrane protein on the surface of the small extracellular vesicles specifically binds to C@MOF@Paper, so that the small extracellular vesicles are captured at the test point. After incubation and washing, the detection probe DP is added to the Exopp-PAD through a syringe. After sufficient reaction, Hemin is added to the Exopp-PAD through a syringe. After washing, the Exopp-PAD is taken out, and TMB single-component colorimetric solution is added dropwise to each test point and reference point. The color development result is observed and photographed, and then the Hue value is analyzed for quantitative analysis. Fig.15As shown in Figures 16a and 16b, the color of the reference point is basically the same in different types and concentrations of small extracellular vesicles. For small extracellular vesicles of liver cancer cells HepG2 and HuH-7, the degree of color change of the test point increases with the increase of small extracellular vesicle concentration, while liver cell LO2 does not show a significant color change. Subsequently, using the self-calibration property of the reference point color remaining unchanged, a ratio-type Hue value detection signal (R-ΔHue = ΔHue sample / ΔHue reference, ΔHue = Hue after reaction – blank Hue) was constructed, and a standard curve was made using R-ΔHue and the logarithm of the small extracellular vesicle concentration, and its detection limit ( Fig.16 b, Table 2).

[0091] The small extracellular vesicles of different cells showed different membrane protein expressions. 8 pc·mL -1 The image of the multi-channel chip is plotted as a histogram, such as Fig.17 As shown in the figure. The expression of membrane proteins of small extracellular vesicles of each cell type is as follows: HepG2: PTK7>PD-L1>EpCAM>CEA; HuH-7: CEA>PD-L1>PTK7>EpCAM; LO2: All four proteins are lowly expressed. The results of highly expressed proteins in HepG2 and HuH-7 are consistent with recent reports. These results indicate that Exopp-PAD can accurately detect the expression of membrane proteins of small extracellular vesicles of cells in a simpler way.

[0092] Table 2 Linear relationship between R-ΔHue and small extracellular vesicle concentration

[0093]

[0094]

[0095] 8. Actual sample analysis

[0096] Based on the established detection probe DP and Exopp-PAD combination device, 5 serum samples were analyzed, including 2 healthy subjects (#1, #2) and 3 liver cancer patient samples (#3, #4, #5, from the biological sample bank of the Seventh Affiliated Hospital of Sun Yat-sen University). Fig.18 As shown in a, in samples #1 and #2, the colors of the test points remained almost unchanged, and the expression levels of the marker proteins corresponding to the test points were all low; sample #3 had a higher response to PTK7 and a lower response to PD-L1; sample #4 had a lower response to EpCAM and sample #5 had a lower response to PD-L1.

[0097] The R-ΔHue of these five samples was quantitatively analyzed and a heat map was drawn ( Fig.18b), further supporting the above observations. A comprehensive comparison showed that the expression levels of all four small extracellular vesicle membrane proteins in healthy individuals (#1, #2) were relatively low, consistent with the expression levels of small extracellular vesicle membrane proteins obtained from LO2 cell culture medium, indicating that samples #1 and #2 are most likely LO2 cell subtypes. For sample #4, its small extracellular vesicle membrane protein expression was CEA>PD-L1>PDK7>EpCAM, which was consistent with the expression of small extracellular vesicle membrane proteins obtained from HuH-7 cell culture medium, indicating that sample #4 is most likely a HuH-7 cell subtype. For sample #3, its small extracellular vesicle membrane protein expression was PTK7>>EpCAM≈CEA>PD-L1, while the small extracellular vesicle membrane protein expression of sample #5 was EpCAM≈CEA>PTK7>PD-L1. This feature is not similar to those cell subtypes we measured, so samples #3 and #5 may belong to other cell subtypes rather than HepG2 or HuH-7. In conclusion, Exopp-PAD has promising application in the non-invasive diagnosis and subtype classification of HCC.

[0098] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A multi-channel paper chip device, characterized in that: The core components of the device are a variety of paper chips C@MOF@Paper modified by aptamers, wherein the C@MOF@Paper includes MOF@Paper on which UiO-66-NH2 is grown in situ on filter paper and nucleic acid aptamers, which are used to specifically recognize small extracellular vesicle membrane proteins.

2. A multi-channel paper chip device according to claim 1, characterized in that: The preparation method of the device is as follows: a plurality of holes are opened on an endurance board, different C@MOF@Paper is filled into each hole as a test point, and MOF@Paper without aptamer modification is filled as a reference point, and then the endurance board is coated with a filter membrane, and then it is loaded into a replaceable membrane filter, and then both ends of the filter are connected to a syringe to prepare a multi-channel paper chip device.

3. A multi-channel paper chip device according to claim 1, characterized in that: The nucleic acid aptamers include the EpCAM aptamer shown in SEQ ID No: 2, the PTK7 aptamer shown in SEQ ID No: 3, the CEA aptamer shown in SEQ ID No: 4, and the PD-L1 aptamer shown in SEQ ID No:

5.

4. A multi-channel paper chip device according to claim 1, characterized in that: The preparation method of MOF@Paper comprises the following steps: S1. Dissolve ZrCl4 in a mixture of formic acid and ethanol, then place filter paper in the resulting mixture and let it stand to allow the surface of the paper to fully absorb Zr 4+ ion, S2. After dissolving BDC-NH2 in a mixture of formic acid, ethanol and water, the BDC-NH2 solution is added to the solution of step S1, and the solution is allowed to stand at room temperature on the surface of the paper to generate MOF nanoparticles, which are then washed and dried to obtain MOF@Paper.

5. A multi-channel paper chip device according to claim 4, characterized in that: The filter paper is Whatman No. 4 filter paper.

6. A multi-channel paper chip device according to claim 4, characterized in that: In the mixed solution of formic acid and ethanol, the volume ratio of formic acid to ethanol is 6-9:20; in the mixed solution of formic acid, ethanol and water, the volume ratio of formic acid, ethanol and water is 6-9:20:7-10.

7. A kit for detecting small extracellular vesicle membrane proteins, characterized in that: The kit comprises the multi-channel paper chip device according to any one of claims 1 to 6 and a detection probe DP, wherein the detection probe DP has a nucleotide sequence as shown in SEQ ID No:

1.

8. A kit for detecting small extracellular vesicle membrane proteins according to claim 7, characterized in that: The method of using the kit is as follows: use the syringe at one end of the multi-channel paper chip device to absorb the body fluid sample to be tested, slowly push it, and pull the syringe at the other end at the same time to make the liquid completely flow into the syringe at the other end, and repeat this cycle to allow the sample to be fully combined with the device, and then replace the body fluid sample to be tested with washing solution, DP detection solution, and Hemin solution in turn, and operate in the same way, and finally take out the multi-channel paper chip device, add TMB single-component colorimetric solution to each test point and reference point, observe the color development results, take pictures, and then analyze the Hue value for quantitative analysis.

9. A kit for detecting small extracellular vesicle membrane proteins according to claim 8, characterized in that: The body fluid sample to be tested is a serum sample.

10. A kit for detecting small extracellular vesicle membrane proteins according to claim 8, characterized in that: The quantitative analysis method is: using the self-calibration property of the reference point color remaining unchanged, construct a ratiometric Hue value detection signal: R-ΔHue = ΔHue sample / ΔHue reference, ΔHue = Hue after reaction – blank Hue, and then use R-ΔHue and the logarithm of the small extracellular vesicle concentration to make a standard curve.

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